Assay to determine lrrk2 activity in parkinson&#39;s disease

ABSTRACT

Disclosed are novel phosphorylation sites identified in LRRK2 and associated with Parkinson&#39;s Disease, antibodies that specifically bind to the novel phosphorylation sites, and laboratory and clinical uses thereof.

This application is a continuation of U.S. patent application Ser. No.14/869,709, filed Sep. 29, 2015, which is a division of U.S. patentapplication Ser. No. 14/118,547, filed Feb. 6, 2014, which is a U.S.National Stage entry of International Application No. PCT/US12/38696,filed May 18, 2012, which claims priority from U.S. ProvisionalApplication 61/487,628, filed May 18, 2011, U.S. Provisional Application61/487,639, filed May 18, 2011, and U.S. Provisional Application61/537,463, filed Sep. 21, 2011, the entire contents of each of whichare hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 7, 2015, isnamed 16816720831_ST25.txt and is 4.98 kB in size.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is a progressive neurodegenerative diseaseaffecting 1-2% of the population over 65 years of age. It is estimatedthat the number of prevalent cases of PD world-wide will double by theyear 2030. Currently, there is no cure, early detection mechanism,preventative treatment, or effective way to slow disease progression.The increasing disability caused by the progression of disease burdensthe patients, their caregivers as well as society. Classic neuronalpathological features of PD include the loss of dopaminergic (DA)neurons in the substantia nigra (SN) and the presence of cytoplasmicinclusions known as Lewy bodies. Classical clinical features of PDinclude resting tremor, bradykinesia and rigidity, but the disease isnow know to have wide variety of non-motor features such as autonomicdysfunction and dementia. Although the pattern of neuronal loss in PD iswell characterized, the molecular mechanisms that lead to that celldeath are still unknown. The majority of PD patients suffer fromidiopathic disease with no clear etiology, and approximately 5% ofpatients present with familial PD.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates domain structure of LRRK2 is to scale, with aminoacid residues indicating domain boundaries and phosphorylation sitesindicated.

FIG. 2 depicts proximity ligation assay (PLA) detection of an antigen(e.g., LRRK2).

FIG. 3 depicts ZFN-driven genome editing.

FIG. 4 shows Western blot analysis of LRRK2 N′ phosphorylation sites.

FIG. 5 is a Western blot showing expression of LRRK2 in iPS cells.

FIG. 6 shows brightfield and immunofluorescence microscopy of neuronaldifferentiation in culture, including A) embryoid bodies, B) neuronalrosettes, C) Neuronal rosettes immunostained against Pax6, D) Hoechstcounterstain.

FIG. 7 shows microscopy of neuronal stem cells, including A) phaseconstrast image of NSCs, B) NSC immunostained against Nestin, C) NSCstained with Soxl, D) differentiated neurons at day 30 stained withgamma-tubulin type III and costain for TH, and E) neurons stained foralpha synuclein and co-stained with TH.

FIG. 8 is a table listing antibodies against total LRRK2 protein orphosphorylated LRRK, and their sources.

FIG. 9 illustrates a sample matrix experiment for testing of total LRRK2Assay Probes.

FIG. 10 shows distribution of kinases across the kinome that modify theindicated residues.

FIG. 11 shows disruption of the G2019S allele (sequence indicated in A)in B) K562 cells and C) patient-derived fibroblasts.

FIG. 12 displays the results of quantitative RT-PCR analysis showingaverage expression of the indicated stress-response genes in iPS andneurons from G2019S (6c3) and control (5c2) lines. Error bars representstandard deviation of 3+ biological replicates. Expression wasnormalized to GAPDH, CentB3, Eef1A.

FIG. 13 shows Western blot analysis of alpha-synuclein and TH expressionin human dermal fibroblasts (HDF), undifferentiated iPS and neurons atday 60 of a culture protocol, as indicated.

FIG. 14 shows a linear domain map of LRRK2. Amino acid boundaries of thedomains are indicated above domain names (LRR, leucine Rich Repeat; ROC,Ras of Complex; COR, c-terminal of ROC; Kinase and WD40). Pathogenicmutations, non pathogenic mutations, and constitutive phosphorylationsites are indicated.

FIG. 15 shows analysis of LRRK2 pSer955 and pSer973 phosphorylation. Theindicated variants of full-length GFP-tagged LRRK2 were expressed from astable and inducible locus in HEK293 T-REx cells and subjected toimmunoprecipitation analysis. Immunoprecipitates were subjected toimmunoblot analysis with anti-GFP, and anti-phospho-Ser910 (αpS910),anti-phospho-Ser935 (αpS935), anti-phospho-Ser955 (αpS955), andanti-phospho-Ser973 (αpS973) antibodies. 14-3-3 binding to the LRRK2variants was assessed by 14-3-3 far-western blot analyses. All blotswere co-probed with phospho antibody and GFP (total protein) monoclonalantibody and analyzed by Odyssey® LI-COR analysis; a representativetotal GFP blot is shown. Hsp90 co-precipitation was assessed by αHsp90immunoblotting. Data are representative of at least two experiments andwere also seen with alternatively FLAG-tagged variants of LRRK2.

FIG. 16 shows primary amino acid sequence surrounding Serines 910, 935,955 and 973.

FIG. 17 shows Ser955Ala and Ser973Ala Localization. T-REx cells linesharboring the indicated LRRK2 variants were induced for 24 h with 1μg/ml doxycycline to induce expression of GFP-LRRK2. After induction,cells were exposed to vehicle control (DMSO) or 1 μM LRRK2-IN1 for 90minutes, then fixed with paraformaldehyde and GFP fluorescence wasimaged. Representative fluorescent micrographs of cultures for theindicated phosphosite mutants in the presence and absence of inhibitorare shown, localization analysis was performed at least three times.White scale bar represents 10 microns.

FIG. 18 shows data demonstrating that endogenous LRRK2 is phosphorylatedon Ser955 and 973. A) Endogenous LRRK2 was immunoprecipitated from Swiss3T3 cells with a mouse monoclonal anti LRRK2 clone N138/6 (NeuroMab) andrabbit polyclonal anti-LRRK2 (α100-500) antibody. Controlimmunoprecipitations are with species specific immunoglobulin (IgG).Immunoprecipitates were immunoblotted with MJFF2 (C41). Arrowheadindicates LRRK2 and antibody heavy and light chains are labeled. B)Endogenous LRRK2 was immunoprecipitated from Swiss 3T3 cells treatedwith DMSO control (−) or with LRRK2-IN1 (+) for 90 min.Immunoprecipitates were subjected to immunoblot analysis with theindicated phospho antibodies. Blots were co-probed with phospho antibodyas well as N138/6 monoclonal antibody and analyzed by Odyssey® LI-CORanalysis; a representative total blot is shown.

FIG. 19 shows data demonstrating that LRRK2 does not re-phosphorylateSer955 and 973 in vitro after in vivo dephosphorylation. A) Transientlyexpressed, FLAG-tagged wild-type or Thr1491Ala (T1491A) LRRK2 wassubjected to immunoprecipitation-kinase assay, using anti-FLAG M2agarose, in the presence (+) or absence (−) of ATP. Reaction productswere probed with antiphospho-Ser1491 (αpThr1491) antibodies in thepresence of dephosphopeptide. Blots were reprobed with anti-FLAG (αFLAG)for total protein control. B) Transiently expressed wild-type LRRK2 wasimmunoprecipitated from HEK293 cells treated with DMSO or LRRK2-IN1 toinduce dephosphorylation of the constitutive sites (Treatment cellculture). Immunoprecipitates were extensively washed then subjected toin vitro kinase assay in the presence or absence of ATP, in the presenceor absence of LRRK2-IN1. Reaction products were immunoblotted withantiphospho-Ser1491 (αpThr1491), anti-phospho-Ser910 (αpS910),anti-phospho-Ser935 (αpS935), anti-phospho-Ser955 (αpS955), andanti-phospho-Ser973 (αpS973) antibodies.

FIG. 20 shows a gel from which quantitative mass spectrometry (MS)14-3-3 was identified as an LRRK2 interactor. HEK-293 cells stablyexpressing GFP, wild-type full-length GFP-LRRK2 or full-lengthGFP-LRRK2(G2019S) mutant were cultured for multiple passages in eitherR6K4 SILAC medium (GFP-LRRK2) or R10K8 SILAC medium [GFP-LRRK2(G2019S)]or normal ROKO SILAC medium (GFP). Cells were lysed and equal amounts oflysates from GFP and GFP-LRRK2 were mixed. Migration of the LRRK2 bandis indicated with an arrowhead and the GFP band is indicated with anarrow. Molecular-mass markers (kDa) are indicated on the left-hand sideof the gels. The entire lane from each gel was excised, digested withtrypsin and processed for MS. Each sample was analysed with Orbitrap MSand quantified using MaxQuant (version 13.13.10).

FIG. 21 shows phosphorylation of Serines 955 and 973 is dependent onLRRK2 kinase activity.

DETAILED DESCRIPTION OF THE INVENTION

Parkinson's Disease (PD) is a movement disorder characterized bygradually progressing bradykinesia, resting tremor, and posturalinstability with an age-related onset [Gelb et al., Arch. Neurol. 56,33-39 (1999)]. In its typical manifestation, it involves primarily thedegeneration and loss of dopaminergic neurons in the substantia nigra,resulting eventually in severe deficiency of the neurotransmitterdopamine. This type of neurodegeneration involves the formation ofintracellular inclusion bodies (Lewy bodies) [Formo, J. Neuropathol.Exp. Neurol. 55, 259-272 (1996)], which contain the protein synuclein asa major constituent [Spillantini et al., Nature 388, 839-840 (1997);Baba et al., Am. J. Pathol. 152, 879-884 (1998)]. PD can therefore beclassified as a distinct protein aggregation disorder affecting specificsubpopulations of neurons.

Besides classical PD, Parkinsonism-related disorders have been definedwith similar impairment of movement as in PD, but extendedsymptomatology involving also memory and cognitive functions. In suchcases Lewy body formation has spread to cortical areas as well,providing for considerable diagnostic overlap with Dementia with Lewybodies (DLB). Because of the pervasive involvement of synuclein in Lewybody formation, these diverse disorders are grouped under the termSynucleopathies. In spite of this conspicuous association, however, Lewybodies may be more of a classification feature, reporting a specificpathobiochemistry, rather than a direct cause of neurodegeneration[Jellinger, Biochem. Biophys, Acta 2008; Parkinnen et al., ActaNeuropathol. 116, 125-128 (2008)]. On the other hand, the observedcommonalities do suggest that certain forms of Parkinson's Disease withDementia (PDD) are mechanistically related to classical PD. However,there are also forms of PDD with completely unrelated disease biologyinvolving a different form of neurodegeneration based on thepathobiochemistry of the microtubule-associated protein tau (tauopathy),as most clearly exemplified by Frontotemporal Dementia with Parkinsonismcaused by mutations in tau protein on chromosome 17 (FTDP-17) [Hutton etal., Nature 393, 702705 (1998)]. Hence, in view of the evolvingmolecular insights into the basis of these neurological disorders theclassical clinical diagnoses will become more advantageously replaced bydisease-mechanism based classifications, especially if the therapeuticconsequences of diagnosis are increasingly less oriented on symptomrelief but rather on causative treatment strategies. PD can present withan unknown etiology (idiopathic or sporadic PD) or from patients with afamily history of PD (familial PD).

Patients with mutations in the LRRK2 gene generally develop PD withclinical symptoms indistinguishable from idiopathic PD at around 60-70years of age. Mutations in LRRK2 are the most common genetic cause ofparkinsonism. As shown in FIG. 1, the LRRK2 gene encodes a large enzymewith an ankyrin repeat region, a leucine-rich repeat (LRR) domain, aserine/threonine kinase domain, a DFG-like motif, a RAS of complexproteins (ROC) domain, a GTPase domain, an MLK-like domain, a CORdomain, and a WD40 domain.

More than 40 missense mutations have been reported that are locatedthroughout the LRRK2 sequence and several mutants have been shown tosegregate with disease [R1441C/G, Y1699C, G2019S and 12020T]. Of these,the most common mutation is the G2019S substitution, which increaseskinase activity 2-3 fold, and R1441C/G/H, Y1699C and 12020T can inducethe formation of cytoplasmic aggregates.

The cellular processes which are regulated by LRRK2, or the processesthat regulate LRRK2 have yet to be elucidated. One hindrance to theefforts to understand the molecular mechanisms causing PD is the lack ofbiochemical understanding of the events that lead to the loss ofsubstantia nigra neurons and other affected areas. Attribution offamilial PD to mutations in specific genes presents the opportunity fordissecting of the cause of neuronal loss if we can gain an understandingof the perturbed function of the mutated gene products.

LRRK2 contains phosphorylation sites that can be described as eitherautophosphorylation sites or constitutive sites of modification.Autophosphorylation sites have the potential to serve as indicators ofLRRK2 kinase activity, and are generally found in the ROC and COR domainof LRRK2. Constitutive phosphorylation sites hold the potential toprovide reference points for identifying upstream and downstreamsignalling pathways for LRRK2 and are generally localized in a clusterpreceding the LRR domain. Serines 860, 910, 935, 955 and 973/976comprise the most commonly identified sites in this region and these aredepicted in FIG. 1.

Substrate sequence analysis predicts that LRRK2 prefers to modify Thrresidues in the context of a Nictide peptide substrate. Constitutivephosphorylation sites are all serines whereas autophosphorylation sitesare predominantly threonine residues, supporting the idea thatconstitutive sites are not autophosphorylation sites.

LRRK2 interacts with a protein known as 14-3-3, which binds a multitudeof functionally diverse signaling proteins, including kinases,phosphatases, and transmembrane receptors. The name 14-3-3 refers to theparticular elution and migration pattern of these proteins onDEAE-cellulose chromatography and starch-gel electrophoresis. The 14-3-3proteins eluted in the 14th fraction of bovine brain homogenate and arefound on positions 3.3 of subsequent electrophoresis. Perturbed levelsof 14-3-3 protein may be found in the cerebrospinal fluid of patientswith neurodegenerative diseases.

LRRK2 interacts with 14-3-3 isoforms via phosphorylation of Ser910 andSer935. The modification of serines 910 and 935 is likely carried out byan upstream kinase that is indirectly controlled by LRRK2. Treatment ofcells with LRRK2 inhibitors induces dephosphorylation of Ser910 andSer935 and causes LRRK2 to accumulate in cytoplasmic aggregates.Phosphorylation of Ser955 and Ser973 is dependent on LRRK2 kinaseactivity in a manner similar to that of Ser910 and Ser935. Additionally,phosphorylation of Ser955 and 973 is disrupted in the context of severalPD associated mutations that induce LRRK2 aggregation and loss of 14-3-3binding. Phosphorylation of Ser973 exhibits a non-reciprocal dependenceon phosphorylation of Ser910/935. Therefore, Ser955 and Ser973 are twosites of LRRK2 modification that can be utilized as readouts of LRRK2kinase activity.

While preferable embodiments of the invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention.

The invention provides methods for detecting phosphorylation of LRRK2and applications thereof. Various aspects of the invention describedherein may be applied to any of the particular applications set forthbelow or for any other types of diagnostic or therapeutic applications.The invention may be applied as a standalone composition or method, oras part of an integrated pre-clinical, clinical, laboratory or medicalapplication. It shall be understood that different aspects of theinvention can be appreciated individually, collectively, or incombination with each other.

The compositions and methods herein can provide effective means fordetection of phosphorylation of LRRK2 useful for a wide variety ofapplications including, for example, determination of LRRK2phosphorylation status in vitro, in vivo, or in situ. Suchdeterminations can be made in a broad array of biological samples suchas those comprising or derived from a tissue or a populations of cellsisolated from a subject. Cell lines derived from such samples may alsobe of particular use and can be maintained in undifferentiated state ordifferentiated to specific cell types such as neuronal cells or neurons.For example, the present compositions and methods can be used to detectLRRK2 phosphorylation in a population of induced pluripotent stem cells(iPSCs) derived from subjects bearing or not bearing a PD-associatedmutation in LRRK2.

Another aspect of the invention provides methods of elucidatingsignaling pathways involving LRRK2 phosphorylation. Signaling pathwaysinclude proteins or other molecules that contribute to or are affectedby LRRK2 phosphorylation. Such molecules generally include kinases andphosphatases as well as proteins that interact with LRRK2 in aphosphorylation-dependent or phosphorylation-independent manner.Embodiments of the invention provide for methods of determiningrelationships between such molecules to elucidate signaling pathwaysinvolving LRRK2 phosphorylation.

Methods of the invention can also be applied to determine functionalroles of LRRK2 phosphorylation. For example, the kinase activity ofLRRK2 can be studied with respect to its phosphorylation as determinedusing the compositions and methods herein. Embodiments of the inventionare also suitable for studying other functional parameters, such as, forexample, the effect of LRRK2 phosphorylation on autophagy, apoptosis,expression levels of LRRK2 or other proteins, localization of LRRK2 orother proteins, or aggregation of LRRK2 or other proteins.

In some aspects, disclosed compositions and methods are used inconjunction with use of LRRK2 inhibitors and substrates. Methods foridentifying and using inhibitors and substrates of LRRK2 are describedin U.S. Patent Publication Ser. No. 2010/0068742 and PCT PublicationSer. No. WO/2010/031988, each of which is incorporated herein byreference in its entirety for all purposes. Some LRRK2 inhibitors areknown, and have been described in US 2010/0273769 A1 and US 2009/0004112A1.

The compositions and methods herein have a broad spectrum of utility inclinical applications including, for example, diagnosis of PD, prognosisof PD, determination of treatment efficacy for PD, and selection of atreatment regimen for a subject suffering from PD.

Phospho-Specific Antibodies

In one aspect, the disclosure provides an isolated phospho-specificantibody that specifically binds LRRK2 only when phosphorylated at aregulatory site. This subset of embodiments includes, but is not limitedto, antibodies specific for phospho-LRRK2 (Ser910), phospho-LRRK2(Ser935), phospho-LRRK2 (Ser955), and phospho-LRRK2 (Ser973).Phospho-specific antibodies that recognize a single phosphorylation sitecan be used individually, or they can be used in combination with otherantibodies or phospho-specific antibodies.

The term phospho-specific antibody refers to an antibody thatspecifically recognizes and binds to one or more phosphorylated residuesof a phosphorylated substrate molecule. The phosphorylated residue thatis recognized by the specific antibody can be a phosphorylated tyrosine,a phosphorylated serine, a phosphorylated threonine or a phosphorylatedhistidine.

Suitable antibodies may be any intact immunoglobulin molecules orfragments thereof (i.e., active portions of immunoglobulin molecules)that are capable of specifically recognizing and binding to an epitopeof a phosphorylated substrate molecule. The type of antibody that can beused in the inventive methods may be either monoclonal (recognizing oneepitope of its target) or polyclonal (recognizing multiple epitopes).

Phospho-specific antibodies for use in the practice of the assay andscreening methods of the invention may be produced or purchased fromdifferent commercial resources. As will be appreciated by one ofordinary skill in the art, any type of antibody can be generated and/ormodified to specifically recognize and bind to an epitope of a substratemolecule phosphorylated at one or more tyrosine, serine, threonine orhistidine residues.

Methods for producing custom polyclonal antibodies are well known in theart and include standard procedures such as immunization of rabbits ormice with pure protein or peptide (see, for example, R. G. Mage and E.Lamoyi, in “Monoclonal Antibody Production Techniques and Applications”,1987, Marcel Dekker, Inc.: New York, pp. 79-97). Anti-phosphoserinepolyclonal antibodies can, for example, be made using the techniquesdescribed by M. F. White and J. M. Backer (as described in Methods inEnzymology, 1991, 201: 65-67, which is incorporated herein by referencein its entirety).

Monoclonal antibodies that specifically bind to a phosphorylatedsubstrate may be prepared using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture.These techniques include, but are not limited to, the hydroma technique,the human B-cell hydroma technique, and the EBV-hydroma technique (see,for example, G. Kohler and C. Milstein, Nature, 1975, 256: 495-497; D.Kozbor et al, J. Immunol. Methods, 1985, 81: 31-42; and R. J. Cote etal, Proc. Natl. Acad. Sci. 1983, 80: 2026-2030). Monoclonal antibodiesmay also be made by recombinant DNA methods (see, for example, U.S. Pat.No. 4,816,567). Other methods have been reported and can be employed toproduce monoclonal antibodies for use in the practice of the invention(see, for example, R. A. Lerner, Nature, 1982, 299: 593-596; A. C. Nairnet al. Nature, 1982, 299: 734-736; A. J. Czemik et al. Methods Enzymol.1991, 201: 264-283; A. J. Czernik et al, Neuromethods: RegulatoryProtein Modification: Techniques & Protocols, 1997, 30: 219-250; A. J.Czernik et al, Neuroprotocols, 1995, 6: 56-61; and H. Zhang et al, J.Biol. Chem. 2002, 277: 39379-39387).

Techniques developed for the production of chimeric antibodies, theslicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate specificity and biological activity, can,alternatively, be used in the preparation of antibodies (S. L. Morrisonet al, Proc. Natl. Acad. Sci, 1984, 81: 6851-6855; M. S. Neuberger etal. Nature, 1984, 312: 604-608; S. Takeda et al. Nature, 1985, 314:452-454). Monoclonal and other antibodies can also be “humanized”;sequence differences between rodent antibodies and human sequences canbe minimized by replacing residues which differ from those in the humansequences by site-directed mutagenesis of individual residues or bygrafting of entire complementarity determining regions. Humanizedantibodies can also be produced using recombinant methods (see, forexample, GB 2 188 638 B).

Antibodies to be used in the methods of the invention can be purified bymethods well known in the art (see, for example, S. A. Minden,“Monoclonal Antibody Purification”, 1996, IBC Biomedical Library Series:Southbridge, Mass.). For example, antibodies can be affinity-purified bypassage over a column to which a phosphorylated substrate molecule isbound. The bound antibodies can then be eluted from the column using abuffer with a high salt concentration.

Included in the scope of the invention are equivalent non-antibodymolecules, such as protein binding domains or nucleic acid aptamers,which bind, in a phospho-specific manner, to essentially the samephosphorylatable epitope to which the phospho-specific antibodies of theinvention bind. See, e.g., Neuberger et al., Nature 312: 604 (1984).Such equivalent non-antibody reagents may be suitably employed in themethods of the invention further described below. Antibodies provided bythe invention may be any type of immunoglobulins, including IgG, IgM,IgA, IgD, and IgE, including Fab or antigen-recognition fragmentsthereof. The antibodies may be monoclonal or polyclonal and may be ofany species of origin, including (for example) mouse, rat, rabbit,horse, or human, or may be chimeric antibodies. See, e.g., M. Walker etal., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Nat'l.Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)).The antibodies may be recombinant monoclonal antibodies producedaccording to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S.Pat. No. 4,816,567. The antibodies may also be chemically constructed byspecific antibodies made according to the method disclosed in U.S. Pat.No. 4,676,980.

The invention also provides immortalized cell lines that produce anantibody of the invention. For example, hybridoma clones, constructed asdescribed above, that produce monoclonal antibodies to the proteinphosphorylation sites disclosed herein are also provided. Similarly, theinvention includes recombinant cells producing an antibody of theinvention, which cells may be constructed by well known techniques; forexample the antigen combining site of the monoclonal antibody can becloned by PCR and single-chain antibodies produced as phage-displayedrecombinant antibodies or soluble antibodies in E. coli (see, e.g.,Antibody Engineering Protocols, 1995, Humana Press, Sudhir Paul editor.)

Phosphorylation site-specific antibodies of the invention, whetherpolyclonal or monoclonal, may be screened for epitope andphospho-specificity according to standard techniques. See, e.g. Czerniket al., Methods in Enzymology, 201: 264-283 (1991). For example, theantibodies may be screened against the phospho and non-phospho peptidelibrary by ELISA to ensure specificity for both the desired antigen andfor reactivity only with the phosphorylated (or non-phosphorylated) formof the antigen. Peptide competition assays may be carried out to confirmlack of reactivity with other phosphoepitopes on the given Target SignalProtein/Polypepetide.

The antibodies may also be tested by Western blotting against cellpreparations containing the signaling protein, e.g. cell linesover-expressing the target protein, to confirm reactivity with thedesired phosphorylated epitope/target.

In an exemplary embodiment, phage display libraries are used forhigh-throughput production of monoclonal antibodies that targetpost-translational modification sites (e.g., phosphorylation sites) and,for validation and quality control, high-throughput immunohistochemistryis utilized to screen the efficacy of these antibodies. Western blots,protein microarrays and flow cytometry can also be used inhigh-throughput screening of phosphorylation site-specific polyclonal ormonoclonal antibodies of the present invention. See, e.g., Blow N.,Nature, 447: 741-743 (2007).

Specificity against the desired phosphorylated epitope may also beexamined by constructing mutants lacking phosphorylatable residues atpositions outside the desired epitope that are known to bephosphorylated, or by mutating the desired phospho-epitope andconfirming lack of reactivity. Phosphorylation-site specific antibodiesof the invention may exhibit some limited cross-reactivity to relatedepitopes in non-target proteins. This is not unexpected as mostantibodies exhibit some degree of cross-reactivity, and anti-peptideantibodies will often cross-react with epitopes having high homology tothe immunizing peptide. Cross-reactivity with non-target proteins isreadily characterized by Western blotting alongside markers of knownmolecular weight. Amino acid sequences of cross-reacting proteins may beexamined to identify sites highly homologous to the target signalingprotein/polypeptide epitope for which the antibody of the invention isspecific.

In certain cases, polyclonal antisera may exhibit some undesirablegeneral cross-reactivity to phosphotyrosine or phosphoserine itself,which may be removed by further purification of antisera, e.g., over aphospho tyramine column. Antibodies of the invention specifically bindtheir target protein only when phosphorylated (or only when notphosphorylated, as the case may be) and do not (substantially) bind tothe other form (as compared to the form for which the antibody isspecific).

In some embodiments, total LRRK2 antibodies are used for comparison tophospho-specific LRRK2 antibodies. Total LRRK2 antibodies have beenproduced in various organisms including mouse (available for purchasefrom Sigma) and rabbit (available for purchase from Sigma and Enzo LifeSciences), as well as sheep. Phospho-specific LRRK2 antibodies may bereferred to herein by the designation “pSer[X]” where [X] represents aphosphorylated residue recognized by an antibody (e.g., pSer910,pSer935, pSer955, and pSer973).

Use of Phospho-Specific Antibodies

Methods of using antibodies for in vitro, in vivo, ex vivo, and in situanalysis are known. Conventional immunoassays include, withoutlimitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Westernblot or immunoprecipitation assays described in Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990).

Antibodies of the invention can be used to detect phospho-LRRK2 fromhumans. In another embodiment, the antibodies can be used to detectphospho-LRRK2 from primates such as cynomologus monkey, rhesus monkeys,chimpanzees or apes. The invention provides a method for detectingphospho-LRRK2 in a biological sample comprising contacting a biologicalsample with a phospho-specific antibody of the invention and detectingthe bound antibody. In one embodiment, the phospho-specific antibody isdirectly labeled with a detectable label. In another embodiment, thephospho-specific antibody (the first antibody) is unlabeled and a secondantibody or other molecule that can bind the phospho-specific antibodyis labeled. As is well known to one of skill in the art, a secondantibody is chosen that is able to specifically bind the particularspecies and class of the first antibody. For example, if thephospho-specific antibody is a human IgG, then the secondary antibodycould be an antihuman-IgG. Other molecules that can bind to antibodiesinclude, without limitation, Protein A and Protein G, both of which areavailable commercially, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary antibody include, but arenot limited to, various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,(3-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes ¹²⁵I, ¹³¹I, ³⁵S or ³H.

One can use the immunoassays disclosed above for a number of purposes.For example, the phospho-specific antibodies can be used to detectphospho-LRRK2 in cells or on the surface of cells in cell culture, orsecreted into the tissue culture medium. The phospho-specific antibodiescan be used to determine the amount of phospho-LRRK2 on the surface ofcells or secreted into the tissue culture medium that have been treatedwith various compounds. This method can be used to identify compoundsthat are useful to inhibit or activate phospho-LRRK2 expression orlocalization. According to this method, one sample of cells is treatedwith a test compound for a period of time while another sample is leftuntreated. If the total level of phospho-LRRK2 is to be measured, thecells are lysed and the total phospho-LRRK2 level is measured using oneof the immunoassays described above. The total level of phospho-LRRK2 inthe treated versus the untreated cells is compared to determine theeffect of the test compound.

In some embodiments, methods for in situ analysis of LRRK2phosphorylation are provided. Antibodies may be further characterized inthis way using normal and diseased tissues to evaluate phosphorylationand activation status in diseased tissue. Also known asimmunohistochemical analysis or immunohistochemistry (IHC), in situanalysis refers to the process of detecting antigens (e.g., proteins) incells of a tissue section by exploiting the principle of antibodiesbinding specifically to antigens in biological tissues. IHC protocolsare well known in the art; see, e.g., Immunocytochemical Methods andProtocols (second edition), edited by Lorette C. Javois, from Methods inMolecular Medicine, volume 115, Humana Press, 1999 (ISBN 0-89603-570-0)and U.S. Pat. Nos. 5,846,739 and 5,989,838. Briefly, paraffin-embeddedtissue (e.g., tumor tissue) is prepared for immunohistochemical stainingby deparaffinizing tissue sections with xylene followed by ethanol;hydrating in water then PBS; unmasking antigen by heating slide insodium citrate buffer; incubating sections in hydrogen peroxide;blocking in blocking solution; incubating slide in primary antibody andsecondary antibody; and finally detecting using ABC avidin/biotin methodaccording to manufacturer's instructions.

Antibodies may be further characterized by flow cytometry carried outaccording to standard methods. See Chow et al., Cytometry(Communications in Clinical Cytometry) 46: 72-78 (2001).

Proximity Ligation Assay

Compositions and methods of the invention can be applied to conduct aproximity ligation assay (PLA). PLA allows for increasing signalintensity for antibody recognition events by combining the specificityof antibody recognition with the signal amplification and detection ofnucleic acids. Methods using PLA provide advantages in selectivity andsensitivity, aspects that are becoming increasingly necessary whenprobing sample sets that are derived from limited source material.

A schematic diagram of PLA is shown in FIG. 2. PLA enhances proteindetection by antibodies. It does this by translating the interactioninto detectable DNA molecules by employing secondary antibody:DNAconjugates. Species specific secondary antibodies conjugated to uniqueshort DNA strands (PLA probes) are used to detect primary antibodydetection of target protein. When two primary antibodies of differentspecies are used (i.e. sheep anti-LRRK2 and rabbit anti-LRRK2 pSer910),the species specific PLA probes detect the primary antibodies, bringingthe DNA strands of the PLA probes in close proximity. The proximity ofthe probes can be detected by either a subsequent addition of circleforming DNA oligonucleotides. Following ligation, the circle DNA isamplified by rolling circle amplification and is typically used in insitu studies. This localized concentration of fluorescent signal easilydetectible in a fluorescent microscope. Ligations of connectingoligonucleotides are and then and detected using real time PCR.Additionally, this technique can be employed in solution where the PLAsare ligated using a connector oligonucleotide. These ligated PLA probesare then detected using quantitative real time PCR to amplify theconnector region of the annealed probes in close spatial proximity.

PLA can be employed using methods or compositions of the invention invitro, in vivo, ex vivo, or in situ. In some embodiments, PLA isperformed using phospho-specific antibodies of the invention in situ.

Cell Types

Cells for analysis using compositions and methods of the invention canbe derived from nervous tissue including, but not limited to brain stem,cerebrum, cerebellum, corpus callosum, glia, and spinal cord. In someembodiments, cells for analysis are derived from any tissue selectedfrom the group consisting of lung, breast, stomach, pancreas, prostate,bladder, bone, ovary, skin, kidney, sinus, colon, intestine, stomach,rectum, esophagus, blood, brain and its coverings, spinal cord and itscoverings, muscle, connective tissue, adrenal, parathyroid, thyroid,uterus, testis, pituitary, reproductive organs, liver, gall bladder,eye, ear, nose, throat, tonsils, mouth, and lymph nodes and lymphoidsystem.

Cell lines for use with the invention can be derived from a mammaliancell of origin. Suitable mammalian cells of origin include, but are notlimited to, hamster, cattle, primate (including humans and monkeys) anddog cells. Various cell types may be used, such as kidney cells,fibroblasts, retinal cells, lung cells, etc. Among suitable cell linesthe Human Embryonic Kidney cell line (HEK 293) is a common,transfectable cell line capable of high-level gene expression. HEK 293cells may be especially useful for immunoassays with antibodies of theinvention. The 3T3 standard fibroblast cell line can also be suitable.Many cell lines are widely available e.g. from the American Type CellCulture (ATCC) collection, from the Coriell Cell Repositories, or fromthe European Collection of Cell Cultures (ECACC).

Cells can be derived from a subject and reprogrammed into inducedpluripotent stem cells (iPSCs). These cells can then be differentiatedinto various cell types, including neuronal stem cells (NSCs) anddopaminergic (DA) neurons, representing a subject-derived neuronal modelof PD. For example, fibroblasts from patients harboring heterozygous andhomozygous LRRK2 mutation encoding the Glycine 2019 Serine mutation, aswell as age matched controls can be employed. The differentiation ofthese cells to DA neurons represents an experimentally tractable modelof PD in a culture dish. Methods of producing iPSCs are known anddescribed in U.S. Patent Publication Ser. Nos. 2010/0041054,2010/0167286, 2009/0324559, and 2010/0003757, herein incorporated byreference in their entireties.

ZFN-Mediated Genome Editing

ZFN-mediated genome editing can be used in conjunction with compositionsand methods of the disclosure. ZFN-mediated methods of genome editingare known and described in U.S. Patent Publication Ser Nos.2010/0055793, 2011/0086015, 2007/0218528, 2010/0257638, 2007/0218528,and 2009/0117617.

ZFNs can be engineered to introduce targeted DNA double-strand break(DSB) at a locus of interest. ZFNs consist of a zinc finger DNA bindingdomain and the cleavage domain of the FokI restriction enzyme. The DNAbinding domain, which contains a tandem array of 3-6 Cys2-His2zincfingers, is designed to target a 9-18 bp site specified by theinvestigator (each finger recognizes ˜3 bp of DNA). Cleavage of DNArequires dimerization of the FokI domain, which is facilitated bydesigning two ZFNs that bind to adjacent sites (with typically a 5 or6-bp gap between the 2 sites) with the correct orientation (FIG. 3). Theresultant DSB is resolved through either the homology-directed repair(HDR) or non-homologous end joining (NHEJ) pathway, which can beexploited to perform precise base alteration (gene correction) orgenerate small deletions or insertions (gene disruption), respectively.

In some embodiments, ZFN-driven genome editing is used to correct theLRRK2 G2019S mutation in iPS cells. In some embodiments, ZFN-drivengenome editing is used to create the LRRK2 G2019S mutation in iPS cells.ZFN-driven genome editing can be applied to produce any point mutation,insertion mutation, or deletion mutation within the LRRK2 gene. Furtherexemplary mutations that can be created or corrected using thetechnology include, but are not limited to, R1441C/G, Y1699C, 12020T,S910A, S910E/D, S935A, S935E/D, S955A, S955E/D, S973A, and S973E/D.

Alternatively, following cleavage by ZFNs, NHEJ-based repair can lead toefficient re-ligation of the broken ends without the requirement for ahomologous donor; the gain or loss of genetic information that istypically associated with this process frequently leads to frameshiftmutations (gene disruption). For example, the LRRK2 G2019S allele can bedisrupted using the same method.

Kinase Assays

In some embodiments, a kinase assay is used to measure LRRK2 activity.Alternatively, a kinase assay may be used to measure phosphorylation ofLRRK2. A kinase assay measures how much phosphorylation has beencatalyzed by the kinase in a known amount of time. A simple presentlyknown method of doing this is to provide one reactant of thephosphorylation reaction that provides a label which can be measured inthe product (and the labelled product differentiated from the labelledreactant). This is commonly is done by providing radioactively labelledATP, the use of which in the kinase reaction will result inradioactively labelled target peptide. Since the phosphoryl grouptransferred from ATP to the target peptide contains atoms of oxygen andphosphorous, it is theoretically possible to use radioactive isotopes ofany of these atoms as the label. In practice, phosphorous (32P or 33P)is the preferred choice. In addition to direct radioactive labeling,there are indirect labeling or capture methods that exploit antibodiesspecific for the phosphorylated form of the peptide. This approach formsthe basis of both radio-immunoassays and non-radiometric immunoassays.In the latter, the phosphopeptidespecific antibody (or secondaryantibody) may carry an integral enzyme activity, such as horseradishperoxidase, which will allow detection by use of a chromogenicsubstrate, or, the antibody may carry some easily detected fluorophoreor phosphor. If the substrate peptide includes an appropriatefluorophore that does not interfere with its suitability as a substrate,the phosphopeptide specific anti body can be employed in a fluorescencepolarization detection scenario. Since the non-phosphorylated peptidewill have more rapid rotational diffusion compared to phosphorylatedpeptide/antibody complex, these two forms of the substrate (unbound andantibody-bound) are distinguishable upon analysis using polarized light.

As is understood by those skilled in the art, in addition to the kinaseenzyme itself and the necessary reactants, for effective and repeatableassay measurements it is also necessary that the reaction occur in anappropriate media composed of appropriate solvent(s), salts, and variousfactors that facilitate the reaction. Water is the preferred media, butother solvents may be used in whole or more preferably in combinationwith water. These include DMSO, ethanol, and other solvents known tothose of skill in the art as being potentially compatible with enzymaticactivity. Of course, it is preferred that buffers be included in theassay to maintain an appropriate pH range. Useful buffers include HEPES,Tris, MOPS, and the like. The pH is preferably about 6.3-8.3, and morepreferably about 6.8-8.8. In a most preferred embodiment, the pH isabout 7.3. It is important to include certain salts in the reactionmixture for optimal activity. In particular, it is preferred to includeMgCl2 and MnCl2 at appropriate concentrations. Finally, certainsurfactants, cofactors, and the like are preferably included. Amongthese are BSA or other stability-enhancing proteins, as well as EDTA orother heavy metal scavenging compounds.

Different substrates may be used, peptides or whole proteins, which maybe natural or artificial substrates, including the LRRKtide and Nictidepeptides. Also, the autophosphorylation of LRRK2 itself can be used as ameasure of activity. Assays can be conducted at different concentrationsof ATP, which may reflect physiological levels, or may proveadvantageous with a particular assay technology. Other assay componentsmay include co-substrates or regulatory molecules, including but notlimited to GTP or non-hydrolysable analogs thereof. LRRK2 assays maycontain full-length protein, or an active fragment thereof, or variousfusion protein constructs, e.g. containing tags commonly used for theconvenience of purification, or may make use of mutants of LRRK2,preferably those which cause PD.

It is preferable to incubate the kinase reaction of the present assay ata temperature between 10-40° C. More preferably, the reaction occurs atbetween 25-40° C. Because it is preferred and easily achieved, thereaction is best incubated at about 30° C. This incubation period canlast anywhere from a few minutes to a few hours, with a time period of20 minutes to 90 minutes being preferred. In some embodiments, theincubation time is about 40 min, 50 min, 60 min, 70 min, 75 min, 80 min,90 min, 100 min, 110 min or about 120 min.

After running the kinase assay, it is necessary to stop the enzymereaction at a pre-determined time. If the reaction isn't stopped at aprecise known time, then it isn't feasible to compare results from onereaction to another. Of course, any reasonable method of stopping thereaction may be utilized, as long as it doesn't interfere with accuratemeasurement of the kinase reaction results. For example, certaincompounds may be added to the reaction mixture that rapidly denature,degrade, or otherwise disable the kinase enzyme. Such compounds includeTCA, phosphoric acid, SDS, and the like. Alternatively, it may befeasible to heat the reaction mixture to the point where the kinaseenzyme protein is permanently denatured. Such action would requireheating to a temperature of at least about 65° C. Other suitable meansare known to those skilled in the art.

After the kinase reaction is complete, and the kinase enzyme isdisabled, isolation of the labelled target substrate is typicallyrequired. If the target substrate is not isolated, it is typicallyimpossible to distinguish the signal generated by the labelled substratefrom the signal generated by unused labelled reactant. In some cases,however, it is not necessary to isolate the labelled target substrate,and in such cases immediate measurement is performed. For example, incertain cases an assay such as a scintillation proximity assay may beperformed, in which case isolation of the labelled substrate isunnecessary.

Those of skill in the art are aware of many means for isolating thelabelled target substrate from the unused labelled reactant. Typicalexamples include gel electrophoresis, precipitation, filtration,chromatography, immunoprecipitation, and the like. Separation of thelabelled target substrate can also be achieved by TCA precipitation ofthe target peptide.

An excellent method of separation is specific binding of the labelledtarget substrate to a solid support followed by washing away of theunused labelled reactant. For this method, a wide variety of solidsubstrates may be used. Factors to be considered in selecting anappropriate substrate include the adhesion and functional retention ofthe immobilizing receptor, accessible surface area for binding, washconvenience, cost, high-throughput adaptability, etc. Frequently, thesolid substrate will be the wall of the reaction reservoir itself.Preferred substrates maximize signal strength and the signal-to-noiseratio. Exemplary substrates include polystyrene microtiter plates, finefibers, polymeric or silica-based microbeads, etc., preferablypre-activated to provide maximal protein binding. When used, microbeadsare selected by size, range and structure to maximize surface area,filter retention and bead suspension time during the assay incubations.

Once the labelled target substrate is separated from unused labelledreactant, it is necessary to measure the amount of labelled targetsubstrate. The means of making this measurement depend upon the type oflabel used. For radioactively labelled target substrates, the amount ofradioactivity present is measured in any of a variety of means known tothose of skill in the art, including scintillation counting,quantitative autoradiography, densitometry, phosphoimaging, and thelike.

If the target substrate is labelled with a fluorescent label, then theamount of label may be measured by quantitative spectrophotometry,fluorescence/chemiluminescence imaging, or the like.

Other methods for detecting kinase activity are based on separations dueto the charge differences between phosphorylated and non-phosphorylatedproteins and peptides. In these respects, techniques based on gelelectrophoresis and HPLC have, among others, been used. In combinationwith these techniques, spectrophotometric and fluorometric detectionhave been used. Reference is made to International Patent Application WO93/10461 and U.S. Pat. Nos. 5,120,644 and 5,141,852 for descriptions ofmany methods heretofore used for detecting protein kinase activity. Alsoreference is made to Toomik et al., Analytical Biochemistry, 209:348-53(1993).

While not strictly necessary, as a practical matter it is highly usefulto include a series of controls for a kinase reaction. If a testcompound is being added to one reaction mixture, it is important to addan identical volume of a similar composition (absent the test compoundonly) to a control kinase reaction. This will then account for anyalterations in kinase activity caused by solvents, salts, or othercomponents of the solution containing the test compound.

It is also very helpful to include positive and negative control kinasereactions that are likewise as similar as possible to the experimentalreactions, but which contain known modulators of the kinase activity. Bytesting known inhibitors and agonists of the kinase activity alongsidethe unknown test compounds, it is easier to control for unforeseeablefluctuations in kinase responsiveness.

One important aspect of the present invention is its suitability for usein screening for modulators of LRRK2. Because the reaction is simple,performable in small volumes (e.g., on microtiter plates), andreproducible, it is possible to screen huge libraries of compounds anddiscover those that modulate LRRK2 phosphorylation in various manners.Such newly discovered modulators are potential pharmaceuticals forhumans and animals.

Autophagy

Autophagy is a degradative mechanism involved in the recycling andturnover of cytoplasmic constituents from eukaryotic cells. Thisphenomenon of autophagy has been observed in neurons from patients withPD, suggesting a functional role for autophagy in neuronal cell death.Autophagic cell death involves accumulation of autophagic vacuoles (AVs)in the cytoplasm of dying cells as well as mitochondria dilation andenlargement of the endoplasmic reticulum and the Golgi apparatus.Autophagic cell death has been described during the normal nervoussystem development and could be a consequence of a pathological processsuch as those associated with neurodegenerative diseases. The formationof AV can be measured by the accumulation of the autophagosome markerLC3 to AV in discreet foci.

When autophagy is induced, the microtubule-associated protein 1 lightchain 3 (LC3) is processed post-translationally into LC3-I, and then toLC3-II, which associates with autophagosome membranes. Quantification ofthe number of cells with LC3-positive vesicles or LC3-II levels (versusactin) allows for a specific and sensitive assessment of autophagosomenumber in large numbers of cells. Furthermore, as EGFP-LC3overexpression does not affect autophagic activity, the numbers ofEGFP-LC3 vesicles have frequently been used to assess autophagosomenumber. Overexpression of LRRK2 in HEK293 cells and in SH-SY5Y cellsincreases the number of multi-vesicular bodies and autophagiccompartments

Autophagy is another functional pathway that can be studied with respectto altered LRRK2 activity. Wild-type and mutant LRRK2 genotype can bestudied for induction of autophagy or autophagic flux using multipleknown methodologies. Induction of autophagy is accompanied by theaccumulation of LC3 puncta in the cytoplasm of cells and can be easilyvisualized by immunofluorescence microscopy with commercially availableantibodies. Comparing control lines versus LRRK2 mutant lines, it ispossible to evaluate the number of endogenous LC3 puncta per cell usingcomputer software such as NIH ImageJ or WatershedCounting3D. If mutantLRRK2 induces autophagy, these puncta should be more prevalent in themutant lines and be potentially abrogated by LRRK2-IN1 treatment.Inducers of autophagy (nutrient starvation, mTORC1 inhibitors) can alsobe tested in these models to serve as positive controls for the assay,as well as evaluate whether altered LRRK2 kinase activity can negativelyaffect the onset of autophagy. A second measure of autophagy that iscrucial to appropriate evaluation of LC3 puncta data is autophagic flux,which reveals the convergence of autophagic compartments with functionaldegradative compartments. Otherwise, LC3 accumulation couldmisinterpreted as an increase in the onset of autophagy instead of adisruption downstream of autophagy induction. A quenching assay can beused to detect the convergence of autophagic compartments with the latelysosomal compartments. Here, de-quenched BSA is heavily labeled withBODIPY TR-X dye, resulting in self quenching of the fluorophore. This isincubated in the culture medium and will accumulate in lysosomes, whereupon degradation, the fluorophore becomes fluorescent and indicative offunctional lysosomes. Co-localization with LC3 is indicative offunctional fusion of autophagic compartments with degradativecompartments. The accumulation of p62, which should not occur ifautophagy is active, can also be measured. Another parameter used tostudy autophagy is LC3 localization. A tandem green and red fluorescentprotein-conjugated to LC3 can be used to detect LC3 localization.GFP-RFP-LC3 proteins localize to the autophagosome, which fuses with thelysosome to form the autolysosome, where degradation of cellularcomponents occurs. Appropriate progression of autophagy brings theGFP-RFP-LC3 protein to the autolysosome where the GFP signal is quenchedby the low pH. Therefore colocalization of RFP and GFP indicates noautophagy, and red fluorescence after autophagy induction indicatesfaithful maturation of the autophagosome. NSCs can be generated thatstably express this fusion protein using lentiviral methods or standardelectroporation and stable cell cloning. Following differentiation,these models can be subjected to the above analyses to assess the fullimpact of mutant LRRK2 on neuronal survival and autophagy as well as theefficacy of LRRK2-IN1 treatment.

Apoptosis

The term apoptosis is intended to mean the cascade of energy (ATP)dependent events triggered by an apoptosis inducer agent and leading toprogrammed cell death through mechanisms commonly involvingintracellular caspase enzymes; commonly requiring about 12 to about 24hrs.; and commonly involving cell death. In certain embodiments theinvention provides methods for assessing apoptosis prior to cellswelling, fragmentation and/or lysis. Mechanistically, during apoptosisdying cells fragment their DNA and become fragmented themselves intomembrane-bounded apoptotic bodies. The released apoptotic bodies areultimately subject to phagocytosis by immune cells. Where potentiallytoxic products resulting from apoptotic cell death are removed byphagocytes, death of a cell commonly does not result in death ofadjacent cells. Apoptosis is most definitively proven to have takenplace by rescuing dying cells and bringing them back to a condition ofgrowth by addition of an apoptosis inhibiting agent. Apoptosis isrecognized to play a fundamental role in cell development, tissuerenewal; generating and regulating immune responses; and, preventingmalignant transformation. Apoptosis has been implicated in thepathogenesis of an increasing number of diseases and may contribute toneuronal loss resulting from acute insults, such as ischemia, trauma orseizures, infarcts, and certain chronic neurodegenerative diseasesincluding Alzheimer's disease and PD.

Assays for measuring cell apoptosis are known to the skilled artisan.Apoptotic cells are characterized by characteristic morphologicalchanges; including chromatin condensation, cell shrinkage and membraneblebbing, which can be clearly observed using light microscopy. Thebiochemical features of apoptosis include DNA fragmentation, proteincleavage at specific locations, increased mitochondrial membranepermeability, and the appearance of phosphatidylserine on the cellmembrane surface. Assays for apoptosis are known in the art. Exemplaryassays include TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTPNick End Labeling) assays, caspase activity (specifically caspase-3)assays, and assays for fas-ligand and annexin V. Commercially availableproducts for detecting apoptosis include, for example, ApoONE®Homogeneous Caspase-3/7 Assay, FragEL TUNEL kit (ONCOGENE RESEARCHPRODUCTS, San Diego, Calif.), the ApoBrdU DNA Fragmentation Assay(BIOVISION, Mountain View, Calif.), and the Quick Apoptotic DNA LadderDetection Kit (BIOVISION, Mountain View, Calif.).

Clinical Methods

In some embodiments, methods of the disclosure are used to diagnose,theranose, prognose, and/or determine treatment efficacy for a subject.The term “subject” is intended to include organisms, e.g., prokaryotesand eukaryotes, which are capable of suffering from or afflicted with adisease, disorder or condition associated with the activity of a proteinkinase. Examples of subjects include mammals, e.g., humans, dogs, cows,horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenicnon-human animals. In certain embodiments, the subject is a human, e.g.,a human suffering from, at risk of suffering from, or potentiallycapable of suffering from PD, similar forms of Parkinsonism, andsynucleopathies involving Lewy body neurodegeneration. In anotherembodiment, the subject is a cell.

The term “treat,” “treated,” “treating” or “treatment” includes thediminishment, amelioration, or alleviation of at least one symptomassociated with or caused by the state, disorder or disease beingtreated, e.g., PD, similar forms of Parkinsonism, and synucleopathiesinvolving Lewy body neurodegeneration. In certain embodiments, thetreatment comprises the induction of PD or a PD-associated disorder,followed by the activation of the compound of the invention, which wouldin turn diminish or alleviate at least one symptom associated or causedby the PD or a PD-associated disorder being treated. Treatment can bediminishment of one or several symptoms of a disorder or completeeradication of a disorder.

Nonlimiting examples of neurodegenerative disease that may be diagnosedor prognosed by the disclosed methods include Alexander disease, Alper'sdisease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Binswanger's disease, Bovinespongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome,Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntingtonsdisease, HIV- or AIDS-associated dementia, Kennedy's disease, Krabbedisease, Lewy body dementia, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple sclerosis, Multiple System Atrophy, Myastheniagravis, sporadic Parkinson's disease, autosomal recessive early-onsetParkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease,Primary lateral sclerosis, Refsum's disease, Sandhoff disease,Schilder's disease, Schizophrenia, Spielmeyer-VogtSjogren-Batten disease(also known as Batten disease), Spinocerebellar ataxia (multiple typeswith varying characteristics), Spinal muscular atrophy,Steele-RichardsonOlszewski disease, Stroke, Tabes dorsalis, Angelmansyndrome, Autism, Fetal Alcohol syndrome, Fragile X syndrome, Tourette'ssyndrome, Prader-Willi syndrome, Sex Chromosome Aneuploidy in Males andin Females, William's syndrome, Smith-Magenis syndrome, 22q Deletion,and any combination thereof.

EXAMPLES Example 1 Assaying Phosphorylation Status of LRRK2

14 different fibroblast lines heterozygous for G2019S were obtained, ofwhich three lines were derived from unaffected carriers, three were frompatients homozygous for G2019S and 15 were idiopathic lines from age andgender matched controls. iPSC lines were successfully derived using aretroviral system with four factors (OCT4, KLF4, SOX2, cMYC). Theselines were then characterized for pluripotency, differentiationpotential, silencing of transgenes, and were karyotypically normal andformed teratoma in SCID mice. A total of 56 fibroblast cells lines wereselected for iPSC derivation. Patients were ascertained for specificmutations in the SNCA, Parkin, LRRK2, and GBA genes as well as sporadiccases.

Examination of three clonal iPSC lines that were derived byreprogramming fibroblasts from a patient heterozygous for the G2019Smutation showed LRRK2 detection. Using a validated antibody raisedagainst a C′ peptide of LRRK2 (Sheep ?LRRK2 aa2498-2513 S374C16), andusing the established LRRK2 expressing cell line Swiss 3T3 as a positivecontrol, LRRK2 was found to be expressed in these IPSC clones.Therefore, this system provides a useful patient derived model forLRRK2-based Parkinson's diseases and allows for studying LRRK2 usingpertinent, patient derived cells.

Full length, FLAG tagged, wild-type or mutant LRRK2 (as indicated inFIG. 4) was expressed from a stable/inducible locus of HEK293 T-Rexcells (Invitrogen). The data show that the antibodies arephospho-specific and reveal that serines 955 and 973 are regulatedsimilarly to serines 910 and 935. Phosphorylation of serines 955 and 973in the kinase dead and in the inhibitor resistant mutant samplesindicated that these are not autophosphorylation events and that theLRRK2-IN1 inhibitor effects are specific to LRRK2. Additionally,quantitation of these results using the Odyssey system revealed thatserine 973 is modified in a manner dependent on phosphorylation ofserines 910 and 935. This indicates that phosphorylation of Serines 973,910 and 935 are interrelated and dependent on LRRK2 kinase activity.

Example 2 Expression of LRRK2 in IPSCs

Equal amounts of cell lysates from iPSCs derived from patients carryingthe G2019S mutation in LRRK2 as well as Swiss 3T3 cells wereimmunoblotted with anti-LRRK2 (upper) and anti-tubulin antibodies(lower). Three different iPSC clones were used, and are indicated by thenumbers 1, 2, and 3 in FIG. 5. Blots were visualized on a LI-COR Odysseyscanner, and the arrow indicates migration of LRRK2. Tubulin stainingindicates equal loading of cell lysates, and the band indicated by thearrow shows detection of LRRK2. A list of anti-LRRK2 andanti-phoshpo-LRRK2 is displayed in FIG. 8.

Example 3 Neuronal Differentiation of iPSC Lines and Evolving FunctionalPD Related Phenotype

A neuronal differentiation protocol was optimized for obtainingpatients-derived iPSCs. The employed technique used embryoid bodyformation (4 days) followed by attachment of embryoid bodies (6-8 days)(FIG. 6A) and isolation of neuronal rosettes expressing Pax6 (FIGS. 6Band C). The cells were expanded as neural stem cells (NSCs) expressingNestin and Sox 1 (FIG. 7A-C). The NSCs can be passaged andcryopreserved. For dopaminergic differentiation the NSCs were culturedin Neurobasal medium supplemented with sonic hedgehog (Shh) (200 ng/ml)and FGF8 50 ng/ml for 10 days followed by withdrawal of Shh and FGF8 andreplacement of BDNF (20 ng/ml), GDNF (20 ng/ml), and dcAMP (1 mM) for20-25 days. After 30 days, cells showed a high yield of gamma-tubulintype III, a marker for an early neuronal phenotype, and showedexpression of tyrosine hydroxylase (TH) (FIG. 7D). Co-stain of tyrosinehydroxylase (TH) was also detected in 15-20% of the cells as well as ahigh yield of microtubule associated protein 2 (MAP2), a postmitoticmature neuronal marker (FIG. 7E). These cells were further characterizedfor other specific mDA markers (i.e.Pitx3, Nun-1, Lmx1A, AADC, VMAT-2and Girk2). This indicates a high proportion of final neuronal contentfor assaying LRRK2. By representing a genetic background that isimportant for the study of LRRK2, these differentiated cells mostclosely approximate the disease state.

Example 4 Testing of LRRK2 Assay Probes for Use in PLA

PLAs of the disclosure are utilized to detect LRRK2 in differentiated DAneurons that may be generated by iPSC lines derived from subjects withor without Parkinson's disease. A matrix experiment is used to testLRRK2 Taqman Assay Probes for use in PLA. An example matrix is shown inFIG. 9, where labeling of biotin antibody with Oligo A or B is assumedto yield equal activity in the assay. The number of combinations to betested can be determined by the equation: (n2−n)/2+(# ofphospho-antibodies, pAb). In the example shown, n=9 and the total numberof reactions equals 42. The non-redundant combinations are shown ingreen, while the pAbs (ie. 3, 4, 5, 6, 7 and 8—in blue) are testedagainst each other. Following testing, the LRRK2 Assay Probes can beused to detect LRRK2 in differentiated DA neurons.

Example 5 Assay for Intrinsic Kinase Activity of LRRK2 in iPSCs

Using iPSCs derived from subjects with the LRRK2 G2019S genotype,intrinsic kinase activity of LRRK2 can be assessed. LRRK2 from largecultures of iPS cells can be immunoprecipitated using anti-LRRK2antibodies proven to be able to immunoprecipitate endogenous LRRK2.Immune complexes are assayed for kinase activity against the Nictidepeptide substrate, and activity is referenced to control IgG.

Example 6 Assaying LRRK2 Phosphosites as Indicators of LRRK2 Activity

Validated reagents were generated that are capable of detectingphosphorylation events on LRRK2, namely Serines910/935/955/973, whichare all regulated by LRRK2 kinase activity. These reagents are used toexamine the phosphorylation status of LRRK2 Serines910/935/955/973 iniPSCs. For these experiments, two subjects can be investigated pergenotype of control, heterozygous (+/G2019S) and homozygous(G2019S/G2019S), with two iPSC clones per genotype of each subject. Thisyields 4 distinct data points per genotype. For each experiment,parallel cultures are assayed where each culture is treated with theLRRK2 inhibitor at 1 uM, a concentration shown to completely inhibitLRRK2 feedback phosphorylation. Parallel cultures of T-REx LRRK2 celllines expressing FLAG or GFP tagged LRRK2 (cell lines we have in hand)serve as positive controls and cultures treated with LRRK2-IN1 inhibitorserve as negative controls.

LRRK2 phospho-specific antibodies are used in a variety of assays toindicate LRRK2 activity, including immunoblots, immunofluorescence, insitu detection, and in solution detection. These assays are described inA-D below. As with all methods of the disclosure, these assays can beperformed usign iPSCs differentiated to neuronal stem cells,iPSC-derived differentiated DA neurons, and parallel cultures treatedwith LRRK2-IN1 or another LRRK2 inhibitor to induce loss of LRRK2phosphorylation.

A) Immunoblots and in Cell Western Assays in iPSCs or NSCs

iPSCs are liberated from the mouse embryonic fibroblast feeder sublayerusing collagenase. Lysates of iPSCs are immunoblotted with one of thetotal antibodies listed in FIG. 8 and phosphorylation ofSerines910/935/955/973 is assessed with the rabbit phosphoantibodieslisted.

B) Immunofluorescence Microscopy

Phosphoantibody reagents may be applied to higher resolution and spatialanalysis through the use of immunofluorescence microscopy. NSCs arecultured in glass bottom culture dishes and conditions are establishedfor detection of LRRK2 and its phosphorylated species using antibodiesof the disclosure.

Differentiated cultures can also be investigated, in culture usingsimilar glass bottom chamber slides. Antibody reactions are visualizedby fluorescence microscopy, providing data on spatial differences inLRRK2 localization. As a control, T-REx cell lines are also assayed.

C) In Situ Detection of Endogenous LRRK2 or Phosphorylated LRRK2

Utilizing two different antibodies to detect a single antigen adds alayer of specificity to the PLA. Specific detection methodologies fordetection of LRRK2 are developed using an array of LRRK2 antibodies.Using pairs of antibodies directed against two epitopes of LRRK2 (e.g.MJFF rabbit anti-LRRK2 & Sheep anti-LRRK2 100-500) and secondaryantibody reagents from Duolink as the PLA probes, it is possible toestablish the detection of LRRK2 in situ or in NSCs derived from iPSCsusing fluorescence microscopy. NSCs can serve as a starting point. Forcontrols for amplification, 293 T-REx cells expressing a control proteinor LRRK2 in the uninduced and induced state are used. T-REx cellsexpressing a control cell and the uninduced LRRK2 expressing cells serveas controls for no amplification (or low levels of detection), whileT-REx cells expressing LRRK2 serve as positive control for detection ofhuman LRRK2.

Similar techniques are used to detect phosphorylated LRRK2 in situ.Other antibody pairs are employed to investigate the phosphorylationstatus of LRRK2, e.g. Sheep anti-LRRK2 100-500 in combination with theRabbit anti-LRRK2 phosphoSerines 910/935/955/973 antibodies.

D) Detection of Endogenous and Phosphorylated LRRK2 in Solution.

Another application of the PLA to detect targets in solution employssimilar principles but relies on rtPCR detection of the ligated proximalprobes. This amplification of signal is detected by fluorometric PCRinstrumentation. In these experiments, iPSCs or NSCs are used as sourcematerial for detection and probe sets are designed to detect total LRRK2as well as modified LRRK2.

Example 7 Application of Proximity Ligation Assays to Detect LRRK2 inDifferentiated DA Neurons and/or Differentiated Dopaminergic Neurons

Immunofluorescence visualization of LRRK2 and its modifications canvalidate PLA data of LRRK2 expression and phosphorylation. Furthermore,increased sensitivity of PLA allows for a larger dynamic range andhigher signal to noise ratio for assays. PLA is performed using methodsof the invention in situ for LRRK2 expression as well as for LRRK2phosphorylation. TH counterstain is used to confirm DA neuronexpression.

Example 8 Functional Studies of LRRK2 Activity

Using assays to detect LRRK2 as well as its phosphoforms indifferentiated DA neurons, functional aspects can be addressed. Theeffect of LRRK2 inhibitor treatment on a G2019S induced phenotype can beexamined, as can the effect of LRRK2 activity on neuron autophagy,differentiation, and/or survival.

These roles of LRRK2 can be evaluated in controls versus disease modelsusing, for example, iPSC clones for control, +/G2019S and G2019Shomozygous and LRRK2 inhibitor to determine how long-termpharmacological inhibition of LRRK2 impacts the differentiation of iPSCsto the DA neuronal lineage. For the persistent exposure regimen, cellsare treated with 1 uM LRRK2-IN1 from the early stages ofdifferentiation, formation; at the neural progenitor stage; and at day5, 10, 15, 20, 25, and 30. During the differentiation process, media ischanged every other day; at these times, LRRK2-IN1 is replenished in theculture medium. Differences between genotypes of cells can be examinedas well as between cultures exposed to inhibitor or not. Morphologicalchanges like neuritic outgrowth can be compared between the cultures andwith DMSO vehicle control. Neuronal architecture is followed using NIHImage J software, and DA neuronal markers expression is monitored. Usingthe iPSCs as models of existing TH neurons with mutant LRRK2 background,the effect of LRRK2 inhibitor treatment on neuronal survival can bestudied.

Autophagy is another functional pathway that can be studied with respectto altered LRRK2 activity.

Wild-type and mutant LRRK2 genotype can be studied for induction ofautophagy or autophagic flux using multiple known methodologies.Induction of autophagy is accompanied by the accumulation of LC3 punctain the cytoplasm of cells and can be easily visualized byimmunofluorescence microscopy with commercially available antibodies.Comparing control lines versus LRRK2 mutant lines, it is possible toevaluate the number of endogenous LC3 puncta per cell using computersoftware such as NIH ImageJ or WatershedCounting3D. If mutant LRRK2induces autophagy, these puncta should be more prevalent in the mutantlines and be potentially abrogated by LRRK2-IN1 treatment. Inducers ofautophagy (nutrient starvation, mTORC1 inhibitors) can also be tested inthese models to serve as positive controls for the assay, as well asevaluate whether altered LRRK2 kinase activity can negatively affect theonset of autophagy. A second measure of autophagy that is crucial toappropriate evaluation of LC3 puncta data is autophagic flux, whichreveals the convergence of autophagic compartments with functionaldegradative compartments. Otherwise, LC3 accumulation couldmisinterpreted as an increase in the onset of autophagy instead of adisruption downstream of autophagy induction. A quenching assay can beused to detect the convergence of autophagic compartments with the latelysosomal compartments. Here, dequenched BSA is heavily labeled withBODIPY TR-X dye, resulting in self quenching of the fluorophore. This isincubated in the culture medium and will accumulate in lysosomes, whereupon degradation, the fluorophore becomes fluorescent and indicative offunctional lysosomes. Co-localization with LC3 is indicative offunctional fusion of autophagic compartments with degradativecompartments. The accumulation of p62, which should not occur ifautophagy is active, can also be measured. Another parameter used tostudy autophagy is LC3 localization. A tandem green and red fluorescentprotein-conjugated to LC3 can be used to detect LC3 localization.GFP-RFP-LC3 proteins localize to the autophagosome, which fuses with thelysosome to form the autolysosome, where degradation of cellularcomponents occurs. Appropriate progression of autophagy brings theGFP-RFP-LC3 protein to the autolysosome where the GFP signal is quenchedby the low pH. Therefore colocalization of RFP and GFP indicates noautophagy, and red fluorescence after autophagy induction indicatesfaithful maturation of the autophagosome. NSCs can be generated thatstably express this fusion protein using lentiviral methods or standardelectroporation and stable cell cloning. Following differentiation,these models can be subjected to the above analyses to assess the fullimpact of mutant LRRK2 on neuronal survival and autophagy as well as theefficacy of LRRK2-IN1 treatment.

Example 9 Forced Proximity Probe (FPP) Test and Evaluation of PLA ProbesUsing Recombinant Proteins

Antibodies are labeled with biotin in batches of 5 at a time using 50 ugof antibody, using standard protocols. Biotin-labeled antibodies arethen conjugated, in separate batches, with streptavidin-oligo probes Aand B. One reason for utilizing multiple total LRRK2 antibodies andmultiple different phosphoantibodies is that these reagents may loseantigen reactivity via the biotin labeling process. The multipleantibodies choices are designed to allow for differential sensitivitiesafter labeling as well as to account for potential loss of reactivity.Employing antibodies that recognize multiple epitopes of LRRK2 increasesthe probability of defining antibody pairs that will faithfullyrecognize the protein.

After biotin labeling and oligo conjugation, antibodies are tested inthe FPP test. The FPP test is a crucial evaluation of the suitability oflabeled antibodies to be employed in the PLA. In this test, antibodiesare evaluated for their efficacy, etc. PLA probe antibodies that passthe FPP test are evaluated for their ability to detect total LRRK2 andphosphoLRRK2 when presented as recombinant proteins. Invitrogen hasgenerated epitope-tagged, full length LRRK2, which will be used foroptimization of LRRK2 total protein PLA. For recombinant, phosphorylatedLRRK2, multiple kinases capable of modifying LRRK2 Serines910/935/955/973 were identified. Bacterially expressed, GST tagged LRRK2800-1300 was used as substrate in a screen of 318 kinases across thekinome. FIG. 10 shows the distribution of kinases capable of modifyingLRRK2 serines. In the figure, individual kinases are represented by asingle lines. Kinase names are omitted, but kinase groups are indicated.

Example 10 Screening and Confirmation of PLA Probes with Purified LRRK2and Phosphorylated Recombinant Proteins

Using validated PLA antibodies against the epitope tags of therecombinant proteins (GST), target- and phospho-specific LRRK2 AssayProbes are screened in combination with anti-epitope Assay Probe. Eighttotal LRRK2 Assay Probes tested against each other and the antiepitopeprobe are described in a matrix shown in FIG. 9. Phospho LRRK2 AssayProbes are tested against the anti-epitope probe and the total LRRK2probes shown to drive successful TaqMan protein assay results. The bestcombination of assay probes (including testing the reverse B vs Aoligos) is confirmed in a repeat of the TaqMan® Protein Assay.

LRRK2 is known to be associated with 14-3-3 as well as Hsp90. 14-3-3proteins associate with LRRK2 in a phosphoserine dependent manner andloss of 14-3-3 protein interaction is indicative of inhibited LRRK2kinase activity or the presence of PD associated mutations that inducecytoplasmic aggregation and loss of 14-3-3 interaction. Hsp90association with LRRK2 helps maintain LRRK2 stability, as geldanamycintreatment is known to decrease LRRK2 accumulation. For both 14-3-3 andHsp90, successfully validated total LRRK2 antibodies are tested incombination with 14-3-3 and Hsp90 antibodies. Serial dilution ofrecombinant LRRK2 Protein Standard is used to determine the limit ofdetection of the assay for, e.g., the best pair of probes for totalLRRK2 and each phospho-LRRK2.

Example 11 LRRK2 Proximity Ligation Assay Validation in Cell Lysates

To transition from recombinant protein assays to cell lysates, theefficacy of the PLA probes to detect recombinant or endogenous LRRK2 isevaluated. Lysates from cells that overexpress LRRK2, either from cellscontaining GFP-LRRK2 or cells transduced with BacMam LRRK2-GFP, are usedto screen the PLA Probes. Lysates are generated using LanthaScreen Lysisbuffer (Invitrogen). HEK293 T-REx™ cells expressing amino-terminal GFPtagged WT, G2019S, D2017A, R1441C/G, 12020T, S910A, S935A, S910A/S935A,S955A and S973A LRRK2 are employed. Additionally, lysates from thedopaminergic cell line SH-SY5Y and/or cells transduced with BacMamcarboxy-terminal GFP tagged full length LRRK2-GFP are used. Using twosources of cell lysate (T-REx™ vs. BacMam) lends insight into theeffects of amino- vs. carboxy-terminal fusions of GFP with LRRK2.Moreover, the phosphorylation status of each LRRK2 construct isevaluated, thus yielding valuable confirmative information on thephosphorylation status of these mutant proteins.

PLA is then applied to cell lines that endogenously express LRRK2. Theamount of total LRRK2 target protein and the phosphorylation status isevaluated and compared across different cell lines. Examples of celllines that endogenously express LRRK2 are Raw macrophages, EBVtransformed human lymphocytes and SH-SY5Y cells. A negative control(HEK293 T-REx™ GFP) and positive control (HEK293 T-REx™ GFP-LRRK2) celllysate are included for comparison.

Example 12 Patient Ascertainment and Peripheral Blood Mononuclear CellPurification for Application of Validated PLA Probes to Patient DerivedSamples

An ultimate and crucial application of the PLA validated probes is toassess the phosphorylation status of LRRK2 in patient derived samples. Astudy group of patients has the following inclusion criteria: Diagnosisof idiopathic PD, no atypical signs of parkinsonism, disease duration 3+years, current age 55-75 yrs, both genders, Caucasian. The control groupconsists of age, gender, and ethnicity matched subjects recruitedthrough the Parkinson's Institute which are spouses or other communitymembers. They are not diagnosed with either PD or any otherneurodegenerative disorder and have no family history of these diseases.The clinical assessment of all subjects includes a general neurologicalhistory and examination, including the modified Unified Parkinson'sDisease Rating Scale (UPDRS), Hoehn and Yahr staging, cognitivescreening (MOCA), and Brief smell identification test (B-SIT).Diagnostic criteria are applied by using all available informationsources (UPDRS ratings, other clinical assessments, medical records).The NINDS criteria, which have integrated the key features of otherdiagnostic schema (CAPIT47, UK Brain Bank), are used. This clinicalassessment is supplemented by detailed structured family histories. Athree-generation pedigree and family history provides the necessaryinformation for identifying additional eligible relatives of the family.All data are stored in password protected databases and safetyprecautions are taken to fulfill protection of any human subject asrequired by law.

Three 10 ml-heparin blood tubes are collected for the isolation ofperipheral blood mononuclear cells using Uni-SepMAXI tube (NOVAmed)using standardized percoll based protocol. During sample ascertainment,cells are snap frozen in liquid nitrogen until all samples are collectedfor analysis. DNA is also collected from a portion of the PBMCs forLRRK2 G2019S genotyping, as well as plasma for future follow-up studies.

Example 13 PLA Detection of LRRK2 in PBMC Lysates

A validated PLA is applied to samples derived from patients to establisha protocol that can be applied to patients treated with LRRK2inhibitors. PBMCs are lysed in LanthaScreen lysis buffer supplementedwith protease and phosphatase inhibitors, aliquoted and snap frozen forrepeat analyses. PLA is performed using these cell samples to detecttotal levels of LRRK2 and assess the phosphorylation of LRRK2 as aputative pharmacodynamic marker.

Example 14 Assaying LRRK2 Dimerization and/or Interactions in CellLysates

PLA is performed using lysates from cells that over-express LRRK2 andcells that express endogenous LRRK2 (i.e. SH-SY5Y or Raw macrophages orhuman lymphoblasts). For the interaction assays, PLA probes for 14-3-3and Hsp90 interacting proteins (from Aim 1A) are initially testedagainst full-length recombinant proteins in combination withanti-epitope Assay Probes. These assays are also performed on lysates ofcells expressing epitope tagged LRRK2 or endogenous LRRK2. In this way,a PLA for LRRK2:14-3-3 interaction can be established and used as anadditional marker of LRRK2 kinase activity inhibition or mutationinduced dephosphorylation of Ser910/935. Additionally, since LRRK2 islikely to be constitutively associated with Hsp90, this interactionassay serves as a positive control for protein:protein interactionsinvolving LRRK2.

LRRK2 has been described as a dimer via multiple interaction interfaces.Additionally the dimerization status of LRRK2 changes in the context ofaltered kinase activity and PD associated mutation. LRRK2 total proteinprobes are used to develop a LRRK2:LRRK2 interaction assay. Fordimerization assays, the PLA is performed using various total LRRK2Assay Probes—either the same anti-LRRK2 mAb or pAb labeled with Oligo Aand B, or the best mAb labeled with Oligo A and the best pAb labeledwith Oligo B. Cells useful for these assays are HEK293 T-REx™ cellsexpressing aminoterminal GFP tagged WT, G2019S, D2017A, R1441C/G, 12020Ttreated with or without LRRK2-IN1 as an initial source of LRRK2 enzyme.

Example 15 Utilization of the Novel Nictide Substrate Sequence, inSilico Prediction, and Peptide Substrate Libraries to Identify LRRK2Kinase Substrates

An antibody raised against the phospho-Nictide sequence is used toretrieve immunoreactive proteins from cell lysates (Swiss 3T3 or Rawmacrophages), which are then be tested for their ability to serve assubstrates of LRRK2. Pre-immune antibody serves as a control forimmunoprecipitation and cells pretreated with H-1152 or sunitinib toprevent LRRK2 phosphorylation of proteins serve as control lysates whichdo not contain LRRK2 phosphorylated proteins. Immunoprecipitates areresolved by SDS-PAGE and stained with colloidal blue. Proteins thatappear to preferentially associate with the anti-Nictide antibody fromuntreated lysates are identified by mass-spectrometry and consideredcandidate substrate proteins. For in silico prediction of substrates,empirical data derived from the Positional Scanning Peptide Libraryscreen, from which the Nictide optimal peptide substrate sequence forLRRK2 was derived, is utilized to interrogate sequence databases forproteins that may serve as potential LRRK2 substrates

Utilizing kinase screening services of Jerini Peptide Technologies GMBH(Berlin, Germany) with recombinant LRRK2, LRRK2 substrate sequencepreference can be weighed against novel substrate peptides revealed fromthe screen to reveal likely substrate candidate substrates. A panel of1600 peptides representing kinase activation sites can be screened, aswell as a panel of 2304 peptides representing annotated phosphosites.Substrates identified in the screen are referenced with LRRK2 substratesequence preference and likely substrates are prioritized. This alsoprovides a basis for substrate preference validation.

Example 16 Identification of Kinases or Phosphatases that Modify LRRK2at Ser910/Ser935

Phosphorylation of Ser910 and Ser935 on LRRK2 has been shown to mediatebinding to 14-3-3 on LRRK2, is dependent on LRRK2 kinase activity andregulates subcellular localization. Monitoring these was shown to be auseful means to evaluate LRRK2 inhibitors in cell culture model systems.The kinase activity responsible for modification of Ser910 and Ser935 isnot an autophosphorylation event and is likely through LRRK2 mediatedpositive regulation of a kinase or negative regulation of a phosphatase.Therefore, a prime source to find a likely substrate for LRRK2 is toelucidate the enzymes responsible for modification of these sites.Multiple complementary techniques can be employed to identify the kinaseresponsible for these modifications: pharmacological inhibition of thekinase, phosphorylation of LRRK2 with a panel of candidate kinases,co-immunopurification as well as a siRNA screen to define thephosphatase that regulates Ser910/Ser935.

Using cells expressing the LRRK2 dependent kinase endogenously, eithernon-selective or specific kinase inhibitors are administered againstupstream kinases (e.g. inhibition of PI3K/mTOR) and kinases that haveknown preferences for basic residues at the −3 and −4 position relativeto the phosphorylation site, in order to narrow the possible kinase(s)responsible for the modification of LRRK2 Ser910/Ser935. The LRRK2Ser910/Ser935 phosphorylation sites were subjected to database searchesusing the netphorest algorithms which include most kinase substraterecognition sequences to help direct the search of the upstream kinase.

LRRK2 kinase-inactive protein is still modified at Ser910/Ser935, albeitat a lower level than active LRRK2. This most likely represents a basallevel of modification of these sites in the absence of LRRK2 activationof the kinase. Therefore, as an alternative approach, to avoid LRRK2kinase activity propagating the feedback of kinase activity onSer910/935, inhibitors are evaluated using kinase inactive LRRK2 as asubstrate; therefore the screen can be repeated in this experimentalbackground. For these experiments, cells expressing recombinant,kinase-inactive LRRK2 (T-REx system) are treated with the test inhibitorand total LRRK2 will be immunoprecipitated, then immunoblotted withanti-phosphoserine 910 (pSer910) and anti-phosphoserine 935 (pSer935)antibodies. Kinases targeted by the tested inhibitors that suppressmodification of Ser910 and Ser935 are classified as candidate substratesof LRRK2. Inhibitors are tested to ensure that they do not inhibit LRRK2directly by testing candidate compounds against recombinant LRRK2 invitro. Potential substrate kinases are also considered putative upstreamkinases for LRRK2 Ser910/Ser935.

An LRRK2 kinase or phosphatase can also be identified usingco-immunoprecipitation analysis. The amino-terminus (amino acids 1-1300)and/or the 780-1300 domain can be used as bait. Using cells expressingeither GFP, or GFP fused to amino acids 1-1300 or 780-1300, anti-GFPaffinity chromatography is performed using ChromoTek nanotrap GFP-Binderbeads. Proteins that specifically are enriched in these pull-downs canbe identified by mass-spectrometry and considered candidate substratesof LRRK2. Additionally, utilization of these isolated domains mayincrease likelihood of identifying the interacting partners, as analysisof these domains in isolation may increase the ‘specific activity’ ofinteraction. To further increase the likelihood of identifying the LRRK2kinase or phosphatase, in these experiments and otherco-immunoprecipitation experiments a reversible chemical crosslinkingagent can be added to the lysis buffers in order to increase theprobability of capturing more transient associations. Thephosphorylation sites in question are close to the leucine rich repeatdomain, which could serve as a protein:protein interaction domain thatbridges the upstream enzyme to the sites of modification and it maytherefore be possible to co-precipitate the modifying enzymes thatregulate these sites.

In vitro, phosphatases may more readily dephosphorylatenon-physiological substrates. Reverse genetics can be used to elucidatethe phosphatase that dephosphorylates Ser910/Ser935, by using a siRNAlibrary targeted against the phosphatase complement of the human genome.The T-REx expression system can be employed to express GFP tagged LRRK2.LRRK2 is localized in a diffuse, cytoplasmic pattern. In the presence ofInhibitor, LRRK2 is found to be dephosphorylated and in cytoplasmicaggregates. For the screen, LRRK2 dephosporylation is assayedimmunologically (loss of signal) or by the induction of GFP-LRRK2aggregates, two solid readouts of LRRK2 modification. First, cells areadministered the phosphatase targeting siRNAs, followed by acute, 90minute, LRRK2 inhibitor treatment. If the phosphatase that mediates thedephosphorylation of LRRK2 is repressed, then LRRK2 inhibition shouldnot result in dephosphorylation and aggregation. GFP fluorescence canserve as an internal control for LRRK2 expression and Ser910Ala andSer935Ala substitution mutants can be used as controls for nonphosphorylated LRRK2. Positive hits of the screen are validated bytargeted siRNA depletion of the phosphatase, followed by biochemicalvalidation that it is a substrate of LRRK2. Utility of this screen isvalidated by utilizing broad spectrum phosphatase inhibitors such ascalicylin A and okadaic acid as hallmarks of phosphatase inhibition.

Example 17 Biochemical and Chromatographic Purification of the LRRK2Kinase

Since cells expressing LRRK2 contain the kinase that modifiesSer910/Ser935, chromatographic techniques are useful to purify thekinase from cell lysates and/or brain lysates. Multiple methods of celllysis can be employed in order to preserve the kinase activity, namelymechanical, isotonic disruption of cells versus detergent basedextraction of cells, which may easily disrupt a potential signalingcomplex (such as the mTOR complex being disrupted by Triton X-10019).The Ser910/Ser935 kinase activity can be enriched by columnchromatography, where cell lysates are fractionated first bydifferential centrifugation (in the case of mechanical disruption) andthen by ion exchange column chromatography. Serine910/935 kinaseactivity is monitored across the column fractions by using portions ofthe fractions as the source of kinase in a kinase assay with recombinantLRRK2 as a source of substrate and monitoring for specific modificationof 910/935 by immunoblot analysis. Fractions containing the activity arefurther enriched until it can be identified by mass spectrometry.

Example 18 Mechanistic Dissection of LRRK2 Phosphorylation

Reagents can be generated to detect phosphosphorylation of serines 860,955 and 973/976 and determine if they are responsive to LRRK2 kinaseactivity. To understand the role of these sites in LRRK2 biology,phosphospecific antibodies are raised against these phosphosites. Theability of these sites to be phosphorylated in response to LRRK2activity will be examined as the 910 and 935 sites have been. Thephosphorylation status of these sites will also be analyzed in the PDassociated mutations and the impact on LRRK2 stability/aggregation willbe assessed.

The carboxy-terminal domain of LRRK2 is necessary for kinase activity,and a detailed study of this domain can help to understand the role thecarboxy terminus plays in regulating LRRK2. For example, the ability ofthis domain to reconstitute an active complex in trans can be determinedby transfection of an LRRK2 variant lacking these 8 amino acids (CΔ8),into cells stably expressing the carboxy terminal domain with or withoutthe carboxy terminal 8 amino acids. After co-expression, activity of theLRRK2 CΔ8 will be assessed by ability to phosphorylate Nictide inimmunoprecipitation kinase assays. To determine if the carboxy terminal8 amino acids can mediate this regulation of LRRK2 alone, lysates ofcells expressing wild type or CΔ8 LRRK2 will be prepared in the presenceof excess CΔ8 peptide encompassing the last eight amino acids; a nonrelated peptide of similar length will used as a control. If kinaseactivity is modulated by this peptide alone it could inhibit wild typeor activate CΔ8. If wild type LRRK2 is inhibited, it will indicate thatthis peptide can compete for a crucial binding site on the activatingprotein, precluding association and activation of the kinase domain. Ifthe CΔ8 is activated by addition of the peptide, it will indicate thatthe C′ domain acts as an autoregulatory domain conferring activity tothe protein. Additionally, this peptide can be added directly to animmunoprecipitation kinase assay LRRK2 (wildtype versus CΔ8) todetermine if it can activate or inhibit the protein in isolation. Ifthis peptide does modulate kinase activity of LRRK2, mutational analysisof the peptide will be carried out to determine the necessary residuesthat confer or inhibit activity.

The carboxy terminus may interact with a protein that confers activityto LRRK2. These proteins can be identified by co-immunoprecipitationanalysis using the carboxy terminal domain of LRRK2 fused to GFP, withand without the last eight amino acids to define specificity. Areversible chemical crosslinking agent to the lysis buffers can increasethe probability of capturing more transient associations. Candidatesubstrates from Nictide based identifications can be prioritized basedon similarity to the Nictide sequence, gene expression patterns andpublished phosphosites databases (e.g. phosphoELM and phosida). To testthe ability of LRRK2 to phosphorylate candidate proteins, cDNAs ofcandidate proteins are cloned and recombinant protein expressed inprokaryotic or eukaryotic cells. It can then be tested whetherrecombinant wild type and G2019S LRRK2, but not kinase inactive LRRK2can phosphorylate these substrates. As a control equivalent molarconcentrations of the C-terminal domain of moesin, which is efficientlyphosphorylated in vitro by LRRK2 at Thr558, can be used. A physiologicalLRRK2 substrate would be phosphorylated by LRRK2 at least as efficientlyas moesin.

When substrate(s) are verified in vitro, site(s) can be mapped using acombination of mutational and phosphoproteomic techniques commonly usedin the field of signaling. These include mass-spectrometricidentification of phosphopeptides or isolation of the phosphorylatedpeptide by enzymatic digest followed by HPLC and direct sequencing byEdman degradation or mass spectrometry. Phosphospecific antibodies canbe used to establish whether the same sites on the substrate arephosphorylated in cells, and how this phosphorylation is affected byoverexpression of wild type or G2019S or kinase-inactive LRRK2, and ifadministration of LRRK2 inhibitors blocks phosphorylation at thesesites. siRNA knockdown of endogenous LRRK2 in Swiss3T3 or Raw cells canbe tested for impact on phosphorylation of the identified LRRK2substrate.

Example 19 Assaying LRRK2 Activity in Human Biological Samples A)Establishing a Method for Directly Assaying LRRK2 in Blood Samples.

Understanding if these assays will work in patient samples is crucial toincluding them as a readout of LRRK2 inhibition as part of a clinicaltrial protocol. The Ser910/Ser935 phosphorylation assays can be appliedto lymphocytes purified from blood samples to establish the assay.Antibody based and density centrifugation based lymphocyte purificationmethods can be compared for ease and rapidity in cell isolation. Cellsare rapidly lysed and Ser910/935 phosphorylation is determined byimmunoprecipitation of LRRK2 followed by immunoblot with Ser910/Ser935antibodies. Additionally, LRRK2 immunoprecipitation kinase assays can beapplied to these samples.

B.) Application of LRRK2 Activity Assays to PD Patient Derived SamplesAdapted to Cell Culture Model Systems.

LRRK2 kinase activity and phosphospecific antibody based readouts ofLRRK2 activity can be applied to Parkinson's disease patient samplesthat have been adapted to cell culture model systems. These systems arevital to establishing drug efficacy or toxicity for clinical trials.Evaluated samples can include control, as well as mutation induced PD(LRRK2 G2019S or other genotype, alpha-synuclein multiplication, andalso early onset PD and GBA mutation, all of which are available). Fromthese disease backgrounds, skin fibroblasts, skin fibroblasts induced tobe iPS cells and lymphocytes can be examined.

LRRK2 activity can be measured in fibroblasts and in lymphocytes,indicating that these could be good model systems to perform initialbiochemical validation of LRRK2 responsiveness to novel inhibitors orwhether newly identified substrates are responsive to LRRK2 activatingmutation. A large library of tissue samples (particularly fibroblasts)from LRRK2 patients is available, including both heterozygous andmonozygous patients. These cell systems are initially characterized bydetermining the phosphorylation status of Ser910/Ser935, or otherphosphorylation sites. The specific kinase activity of endogenous, wildtype LRRK2 versus LRRK2 from samples harboring mutations is alsodetermined by immunoprecipitation kinase assay against the Nictidesubstrate. These assays yield insight into the steady state level ofLRRK2 activity.

iPS cells derived from PD patients harboring LRRK2 and alpha synucleinmutations can also be tested. These cells continuously replicate and area regenerative source of mutant LRRK2 model systems. The steady statelevel activity of LRRK2 can be characterized from these cells in theundifferentiated state, as well as cells differentiated to a neuronalstate. An alternate technique of visualizing phospho Ser910/Ser935 canbe used, whereby immunofluorescence microscopy of differentiated neuronsis employed using phosphospecific antibodies against these sites, todetermine a difference in kinase activity between the differentiated andundifferentiated state.

Example 20 Characterization of LRRK2 Stability and Aggregation and Linkto Autophagy

Two apparent general classes of PD associated mutations have beenobserved. One class of mutation affects kinase activity, eitherpositively or negatively. The other confers an increased propensity toaggregate, which is likely to be caused by a reduction in the ability tobind 14-3-3, while not affecting kinase activity in immunoprecipitationkinase assays. Aggregates formed by PD associated mutation can be usedto dissect the role of LRRK2 in signaling pathways. The tetracyclineregulatable (tetR) system allows for the distinction between aggregatesinduced by pathogenic mutation when compared to wild type LRRK2expression. Using the inducible tetR system, potential disruption ofconstitutive autophagic process may be avoided.

LRRK2 is found in aggregates when examined in the context of certain PDassociated mutations, acute pharmacological inhibition and disruption of14-3-3 binding. Wild type LRRK2 expressing cells are treated withinhibitors of, and siRNAs against a) the ubiquitin-proteasome pathway b)the aggresome formation pathway c) the autophagy pathway and d) cellularchaperone apparatus and are evaluated microscopically for the formationof wild type LRRK2 aggregates. Conversely, cellular factors mayfacilitate aggregate formation in the context of certain LRRK2mutations. Cells expressing LRRK2 aggregation prone mutants are screenedwith siRNAs and drugs for the ability to dissipate the aggregates. PDassociated mutations may confer an intrinsic instability to apolypeptide that self associates and therefore possess a higherpropensity to aggregate if small protein synthesis imbalances occur.Alternatively, these mutations may disrupt an as yet uncoveredinteraction with one of these cellular processes which in turn resultsin a disruption of LRRK2 intracellular localization. Utilizing theSer910Ala/Ser935Ala mutants, colocalization experiments are conducted todefine the components of LRRK2 aggregates.

Components of the cytoskeleton as well as vesicle trafficking pathwayscan be examined. Alternatively, LRRK2 can be immunoprecipitated whenexpressed as aggregation prone mutations in order to identify by massspectrometry, novel proteins that preferentially associate with theaggregating LRRK2 versus wild-type LRRK2.

14-3-3 binding to LRRK2 is sensitive to LRRK2 kinase activity; however,the binding of 14-3-3 does not affect protein kinase activity. LRRK2that is unable to be modified at Ser910 and Ser935 (i.e. Ser910A andSer935A) exhibits altered localization compared to wild type.Additionally, acute administration of LRRK2 inhibitors results in theaggregation of LRRK2. It can be determined if overexpression of 14-3-3suppresses the aggregation of LRRK2 induced by mutation. If overexpression of 14-3-3 does relieve the formation of aggregates, acellular auto-protective mechanism will have been revealed where LRRK2kinase activity promotes association of a cellular factor that preventsaggregate formation.

Aggregation prone mutations can be screened in the context of a kinaseinactive mutation, i.e. R1441G/C/H+D2017A, Y1699C+D2017A, etc., for thetendency to form aggregates. It has been shown that proteinaciousaggregates induced by pathogenic mutation in the Huntington protein(polyglutamate expansion HttQ103), the Ataxin1 (polyglutamate expansionAtxQ82) or rhodopsin (P23H mutation) all disrupt the ubiquitinproteasomesystem (UPS) and cause accumulation of reporters of UPS dependentprotein degradation. This UPS monitoring system is applied to cellsexpressing LRRK2 wild-type or LRRK2 aggregation prone mutations. IfLRRK2 aggregation prone mutations induce a global disruption of the UPS,it reveals a pathway of LRRK2 pathogenesis possibly crucial inunderstanding disease.

Direct assessment of LRRK2 protein:protein interaction with componentsof the autophagy machinery will be carried out usingco-immunoprecipitation analysis. It will be determined whether autophagycomponents serve as direct kinase substrates of LRRK2 in vitro, and ifso, the impact of these modifications exert on their role in theautophagy process will be determined in vivo by employing phosphositemutants (unphosphorylatable Ala or phosphomimic Glu) and monitoring theeffects on constitutive autophagy or on autophagy after induction bystarvation or bafilomycin A treatment. Expression of the LRRK2 PDassociated mutants, especially ones which induce protein aggregation,will be evaluated for their affect on autophagy by measuring conversionof LC3I to LC3II, effects on p62 levels and formation of LC3intracellular puncta under normal and autophagy induction conditions inaccordance with the guidelines for monitoring autophagy.

LRRK2 is reported to be ubiquitinated by CHIP and this regulates thelevel of protein stability. It will be assessed whether this is a signalfor chaperone mediated autophagy by evaluating whether co-expression ofLRRK2 and CHIP, leads to enhanced ubiquitination of LRRK2, increasinginteraction or expression of p62, the protein which binds ubiquitinatedproteins and LC3 to link with autophagy.

Example 21 Generation of iPSC Lines from Parkinsonian Patients with theLRRK2 G2019S Mutation and Unaffected Mutation-Negative Controls

iPS cell lines are derived from PD patients carrying mutations indifferent PD genes and controls. Fibroblasts have been derived from skinbiopsies and banked from 14 patients who are heterozygous for the LRRK2G2019S mutation. Fibroblast cell lines have been developed from threepatients who are homozygotes for the mutation. Using a retroviral systemto deliver four genes encoding OCT4, KLF4, SOX2 and cMYC, iPSC lineshave been successfully derived from five LRRK2 patients and nine controliPS lines. These lines have been characterized for pluripotency and arekaryotypically normal. They are further assessed for teratoma formationand promoter methylation of Oct4 and Nanog.

Example 22 ZFN-Mediated G2019S Allele Disruption in Patient-DerivedFibroblasts

FIG. 11 shows efficient cleavage of the LRRK2 gene in a pool ofpatient-derived fibroblasts that were transfected with ZFN. Thetransfected cell pool was subject to single cell cloning—clones thatcontain either insertion or deletion at the site of DSB will lose theBsrDI restriction site. Sequencing analysis of 7 such clones revealed 3clones that contain frameshift mutations of the G2019S allele, thusdisrupting the translation of the protein.

Referring to FIG. 11, the asterisk (*) denotes the base change thatresults in G2019S mutation, ZFN binding sites are underlined. ZFP drivesLRRK2 gene modification in K562 cells (B) and patient-derived (G2019Sheterozygotes) fibroblasts (C). A 346-bp region encompassing the ZFNcleavage site was PCR-amplified from ZFN-transfected cell pools,generating a mixture of unmodified as well as modified amplicons(derived from NHEJ-mediated imperfect repair of the DNA break);denaturing and reannealing of this mixture results in mismatches betweenheteroduplexes of the unmodified and modified alleles, which createdistortions that are recognized and cleaved by Cel-1 nuclease. The topband represents uncut PCR products, the cleavage products are indicatedby #. For patient-derived fibroblasts, * denotes cleavage productsresulted from the heteroduplex between wt and unmodified G2019S alleles.The relative intensity of the cleavage products compared with theparental band was quantitated by densitometryis, and provides a measureof the frequency of ZFN-mediated cleavage of the LRRK2 gene that wasrepaired by NHEJ—˜19% in K562 cells and 16% in fibroblasts.

Example 23 Stress Response of Neurons Derived from G2019S and ControlCell Lines

To further increase the yield of midbrain dopaminergic (mDA) neurons,iPSC-derived neurons are cultured under low oxygen conditions (3-5%oxygen). Referring to FIG. 12, NSCs and TH-positive neurons were derivedfrom normal and LRRK2 positive clonal iPSC lines. These cells werefurther characterized for other specific mDA markers (i.e. Pitx3,Nurr-1, Lmx1A, AADC, VMAT-2 and Girk2).

During a time course the mDA neurons from a patient carrying the LRRK2G2019S on both alleles showed an increase in markers of stress responsegenes as well as an increase in alpha-synuclein protein expressioncompared to control lines (FIG. 13) exhibiting an early functionalphenotype, both of which could be characteristic of PD pathologyMitochondrial function and lysosomal abnormalities can be investigatedin these iPS-derived mDA neurons.

Example 23 Generation of Patient-Derived iPSCs with Corrected orDisrupted LRRK2 G2019S Allele

Patient-derived iPS cell lines with corrected or disrupted G2019S alleleprovide cell models and their corresponding isogenic controls are usefultools. The G2019S allele is disrupted to achieve a functional knockdown,because the G2019S mutation is a gain-of-function mutation and shows anat least a two to three-fold increase in kinase activity due to anincrease in reaction rate. Patient-derived iPSCs with corrected ordisrupted G2019S allele provide cell models to determine whether theG2019S mutation is necessary for developing PD-related phenotypes, andhelp to understand the relative contribution of the G2019S to PDphenotype. Correction and disruption of the G2019S mutation is performedin a minimum of two independent iPSC lines derived from the samepatient; and the same process is carried out on iPSCs derived from 3unrelated patients that carry the mutation, providing isogenic cellmodels for studying the role of G2019S mutation in the context ofdifferent genetic backgrounds.

A lead ZFN pair (LRRK2 ZFNs) that cleaves DNA at a site ˜20 bp upstreamof the G2019S mutation has been validated in K562 cells andpatient-derived fibroblasts. The DSB introduced by ZFNs can be repairedvia HDR or NHEJ-based pathways, resulting in either correction ordisruption of the mutant allele.

Plasmids encoding LRRK2 ZFNs, the green fluorescent protein (GFP) and adonor DNA construct will be co-delivered by nucleofection topatient-derived iPSCs that are heterozygous for the G2019S mutation(generated under CIRM grant TR1-01246). The donor construct contains 1kb of wild type (wt) LRKK2 genomic sequence (500 bps in each directionof the mutation). In a subpopulation of cells that contain ZFN-mediatedDSB, HDR uses the donor as a template to repair the DSB as well as themutation; the majority of the cells use NHEJ to resolve the DSB.Transfected (GFP-positive) cells are enriched through fluorescence-basedcell sorting and replated on MEF feeder layers for single-cell cloning.Genomic DNA can be isolated from 200-250 clones, the region of interestamplified by PCR and subjected to digest by restriction enzymes SfcI andBsrDI. SfcI only cleaves unmodified G2019S allele; while BsrDI site isdestroyed if deletion or insertion has occurred at the site of DSB(suggesting NHEJ). Single cell-derived clones that contain 2 wt alleles(gene correction) will lose the SfcI site and retain BsrDI site on bothalleles, while clones that contain intact wt allele and disrupted G2019Sallele will retain BsrDI on one allele. Clones meet these criteria willbe subject to sequencing analysis to confirm the presence of 2 wt LRRK2alleles for G2019S corrected cells, and to identify those haveframeshift mutations on the mutant allele and unmodified wt allele.Before further functional characterization of gene-corrected or G2019Sdisrupted iPSC clones, cytogenetic analysis will be performed to confirmthey have normal karyotype, and their pluripotent state will be verifiedby expression of the pluripotency markers (OCT4, NANOG and SOX2), aswell as ability to form all three developmental germ layers interatoma-formation assays.

Alternatively, LRRK2 gene modification can be performed inpatient-derived fibroblasts and iPSCs derived from ZFN/donor-transfectedfibroblasts pool. This approach provides an alternative path tocorrected iPS cells, and because fibroblasts are easier to maintain incell culture, they can be transfected with significantly higherefficiency (>90% using nucleofection). Patient-derived fibroblasts arethen nucleofected with LRRK2 ZFN expression vectors and the donorconstruct, cel-1 nuclease assay will be performed on a sub-population oftransfected cells to confirm that efficient cleavage by ZFNs hasoccurred at the LRRK2 locus. The transfected pool is used for iPSCderivation; the resultant iPSC clones subjected to genotype analysis.Clones with 2 wt LRRK2 alleles, as well as those with frameshiftmutation on the G2019S allele and intact wt allele, will be identifiedand their pluripotency verified before additional functionalcharacterization.

Example 24 De Novo Creation of LRRK2 G2019S Mutation in Normal iPS Cells

LRRK2 ZFNs can also be used to introduce DSBs to the LRRK2 locus iniPSCs derived from normal subjects, and HDR can be invoked for de novocreation of monoallelic G2019S mutation; the resultant iPS cell linescan provide powerful evidence whether the G2019S mutation is sufficientfor producing PD-related phenotypes in terms of increased kinaseactivity and ultimately pathological manifestation of PD in dopaminergicneurons derived from iPS cells.

For these experiments, iPS cells from normal subjects are used, and thedonor construct contains the nucleotide that introduces the G2019Smutation. The desired clone will contain SfcI site on one allele andBsrDI on both alleles, clones with the expected digest pattern will besequenced to verify the engineered mutation. Alternatively, anon-integrating lentiviral vector is used deliver both the ZFNs and thedonor to patient-derived iPSCs.

Example 25 Assessment of LRRK2 Protein and Kinase Activity inZFN-Modified Cell Lines

The first functional measures to assess the impact of the ZFN-mediatedgene editing are the direct effect on the LRRK2 protein and LRRK2 kinaseactivity in iPS cells. Immunoblot analysis is used to ensure theZFN-editing does not affect the steady state accumulation of LRRK2 whencompared to wild type cells. The amount of LRRK2 kinase activity inedited iPS cells is determined using the ability to immunoprecipitateand assay endogenous LRRK2 against a novel and specific peptidesequence-Nictide15. The amount of LRRK2 kinase activity is evaluated inthese cells by immunoprecipitation of endogenous LRRK2 from wild-type,G2019S het and ZFN mediated wild-type reversions, followed by assayingthe activity against Nictide in immune complex kinase assays. Anotherassay of LRRK2 kinase activity is based on the demonstration that LRRK2kinase activity potentiates a feedback phosphorylation event on Serines910 and 935. Monitoring these sites can detect the loss of LRRK2 kinaseactivity. LRRK2 kinase activity is monitored using phospho-specificantibodies against phosphoserine 910 and phosphoserine 93516. Otherantibodies targeted to LRRK2 constitutive phosphorylation sites as wellas against autophosphorylation sites may also serve as useful readoutsof LRRK2 kinase activity. Having isogenic control cell lines adds greatprecision to these experiments. Alternatively, in situ orimmunofluorescence techniques of the disclosure can be used, e.g.,examination of phosphoserine 910/935 phosphorylation byimmunohistochemical approaches.

Example 26 Evaluation of a PD-Related Phenotype in ZFN-Modified CellLines in Differentiated Midbrain Dopaminergic Neurons

mDA neurons have been derived that show characteristics of A9 mDAneurons of the substantia nigra. The expression (RT-PCR and IHC) ofother midbrainconsistent markers can be measured in these mDA neurons.Neuronally differentiated iPS cells can be examined for signs ofspontaneous pathology starting with simple measures of mDA cellabundance (% DA neurons after differentiation) and general morphologicalattributes such as inclusion bodies, or dystrophic neuritis andfunctional dopamine release measured by HPLC).

Stress responses can be compared in mDA neurons from the generated panelof ‘repaired’, disrupted, and created G2019S mutation and unmodified‘original’ iPS lines. Several clones from the same lines can be testedas well as lines from unrelated LRRK2 carriers. Parameters to beexamined include molecular signs of pathology of PD, which includebiochemical markers of oxidative stress, a-synuclein overexpression, andmitochondrial and lysosomal dysfunction. Assays for disease-associatedmechansisms include apoptosis (TUNEL, caspase activation), necrosis(CytoTox-Glo), 3) oxidative stress (glutathione, ROS and 4-HNE), 4)mitochondrial dysfunction (ATP-lite: ATP production, membrane potential,mitochondrial content). Protein aggregation of a-synuclein can beassessed with antibodies against total, phosphorylated and nitrateda-synuclein to detect aggregates, inclusion bodies, and neuriticpathology.

Neurons can be sampled for candidate gene profiling at multiple timepoints during the dopaminergic differentiation using a carefullyselected set of neuronal markers as well as markers of specificpathology related to neurodegeneration of PD (oxidative stress genes,mitochondrial genes, lysosomal genes, axon-guidance pathway genes).Alternatively, global comparisons can be made for expression differencesin these cell lines using commercially available microarray platformsfor mRNAs to identify potential downstream targets or microRNAs, sincethere is recent evidence that LRRK2 regulates miRNA-mediatedtranslational repression. The availability of isogenic controls avoidsmany confounding factors in data interpretation.

To further sensitize the disease model of this approach, disease LRRK2cells and corrected cells can be subjected to known toxicants andstressors to see if they exhibit different responses. Possible agentsfor this approach include neurotoxicants such as MPTP/MPP+(aneurotoxicant specific for dopaminergic neurons), 6-hydroxydopaminewhich induces cell death and oxidative stress, and the pesticiderotenone, a mitochondrial toxin.

Example 27 Disruption of Phosphorylation of LRRK2 Serines 955 and 973 byParkinson's Disease Mutations and LRRK2 Inhibition I. Methods A.Reagents and General Methods.

Tissue-culture reagents were from Life Technologies. The Flp-in T-RExsystem was from Invitrogen and stable cell lines were generated as permanufacturer instructions by selection with hygromycin. Restrictionenzyme digests, DNA ligations and other recombinant DNA procedures wereperformed using standard protocols. DNA constructs used for transfectionwere purified from Escherichia coli DH5alpha using Qiagen plasmid Maxikits according to the manufacturer's protocol. All DNA constructs werekindly provided by Dr. Dario Alessi (MRC-PPU, Dundee University, DundeeScotland) except pcDNA5/FRT/TO+GFP-LRRK2-S860A,pcDNA5/FRT/TO+GFP-LRRK2-S955A, pcDNA5/FRT/TO+GFP-LRRK2-S973A,pcDNA5/FRT/TO+GFP-LRRK2-S976A, and pcDNA5/FRT/TO+GFP-LRRK2-5973A/S976Awhich were sub-cloned from the corresponding pCMV5-FLAG constructs.

B. Buffers.

Lysis Buffer contained 50 mM Tris/HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM NaF, 5mM sodium pyrophosphate, 0.27 M sucrose, 1 mM Benzamidine and 1 mMphenylmethanesulphonylfluoride (PMSF) and was supplemented with 1%Triton X-100. Buffer A contained 50 mM Tris/HCl, pH 7.4, 50 mM NaCl, 0.1mM EGTA and 0.27 M sucrose. Cell culture, treatments and cell lysis.HEK-293 cells were cultured in Dulbecco's Modified Eagle's mediumsupplemented with 10% FBS, 2 mM glutamine and 1× antimycotic/antibioticsolution. HEK-293 T-REx cell lines were cultured in DMEM supplementedwith 10% FBS and 2 mM glutamine, 1× antimycotic/antibiotic, and 15 μg/ml

Blasticidin and 100 μg/ml hygromycin. Cell transfections were performedby the polyethylenimine method. T-Rex cultures were induced to expressthe indicated protein by inclusion of 1 μg/ml doxycycline in the culturemedium for 24 hours. Per 15 cm dish, cells were washed once with PBS andlysed in situ with 1.0 ml of lysis buffer on ice, then centrifuged at15,000×g at 4° C. for 15 minutes. Protein concentrations were determinedusing the Bradford method with BSA as the standard.

C. Antibodies

Antibodies against LRRK2 phosphosites were produced by Yenzyme Inc.Phosphoserine 910 antibodies were generated by injection of the keyholelimpet hemocyanin (KLH) conjugated phosphopeptide CLVKKKSNpSISVGE (SEQID NO: 1) (where pS is phosphoserine, Cys added for conjugation) intorabbits and was affinity purified by positive and negative selectionagainst the phospho and de-phospho peptides respectively. Antibodiesagainst LRRK2 phosphoserine 935 were generated by injection of the KLHconjugated phosphopeptide CLQRHSNpSLGPIFDH (SEQ ID NO: 2) (where pS isphosphoserine) into rabbit and was affinity purified by positive andnegative selection against the phospho and de-phospho peptidesrespectively. Antibodies against LRRK2 phosphoserine 955 were generatedby injection of the KLH conjugated phosphopeptide CRKRKILSpSDDSLR (SEQID NO: 3) into rabbit and were affinity purified by positive andnegative selection against the phospho and de-phospho peptidesrespectively. Antibodies against LRRK2 phosphoserine 973/phosphoserine976 were generated by injection of the KLH conjugated phosphopeptideCHMRHSDpSISpSLASERE (SEQ ID NO: 4) into rabbits and were affinitypurified by positive and negative selection against the phospho andde-phosphopeptides respectively. The LRRK2 phosphoserine973/phosphoserine 976 antibody only recognizes the pS973 phosphosite asis shown in FIG. 14. Recombinant LRRK2 100-500 was generated byexpression as a GST fusion protein in bacteria and purification onglutathione sepharose (obtained from Amersham) followed by cleavage ofthe GST moiety with Precission Protease. Antibodies against total LRRK2were generated by injection of LRRK2 100-500 into rabbits followed byaffinity purification against uncleaved GST-100-500. Rabbit polyclonalantibodies against LRRK2 phosphothreonine 1491 were generated by theMIFF against a phospho Thr1491 peptide. Anti GFP monoclonal antibody wasfrom Roche (clones 7.1 and 13.1, #11814460001). The Nanotrap GFP-Trap_Amatrix was from ChromoTek (GTA-20). Anti-FLAG M2 antibody and affinitymatrix were from Sigma (A2220). LiCOR Dyelight labelled 14-3-3 protein(recombinant, His tagged BMH1, a kind gift of Dr. Dario Alessi) wasprepared as per the manufacturer's instructions and used to detect14-3-3 binding. Anti-Hsp90 (heat-shock protein 90) antibody was fromCell Signalling Technology (#4877). Fluorescent secondary antibodieswere from Li-COR (Lincoln Nebr.) or Rockland immunochemicals (RocklandIll.) LRRK2 Immunoprecipitation assays. Cell lysates were prepared inLysis buffer (1.0 ml per 15 cm dish) and subjected toimmunoprecipitation with GFP-trapA beads at 10 ul beads per 1.0 mllysate for 1 hr. Beads were washed twice with Lysis Buffer supplementedwith 300 mM NaCl, the twice with Buffer A. Immune complexes were boiledin Laemelli sample buffer and incubated at 70 degrees C. for 10 minutes.Immunoprecipitation Kinase Assay conditions. Wild-type LRRK2 wastransiently expressed in HEK293 cells using polyethylenimine forapproximately 36 hours, then cells were treated with DMSO or 1 μMLRRK2-IN1 for 90 minutes. Lysates were then subjected toimmunoprecipitation with anti-FLAG M2 agarose at a ratio of 1 μl beadsper 100 μg of lysate for 2 hours. Immune complexes were washed threetimes with lysis buffer supplemented with 300 mM NaCl, followed by threewashes with buffer A. Immune complexes were subjected to in vitro kinaseassay by incubation with 50 mM Tris [pH 7.4], 0.1 mM EGTA, with orwithout 100 μM ATP and in the presence or absence of LRRK2-IN1. Reactionproducts were analyzed by immunoblot analysis.

D. Fluorescence Microscopy.

HEK-293 Flp-in T-REx cells harbouring GFP tagged LRRK2 and PD associatedmutations were plated in 4-well glass bottom, CC2 coated chamber slides(Nunc). One day after plating, cells were induced with 1 μg/mldoxycycline and 24 hr later, cells were fixed in 4% paraformaldehydebuffered in phosphate buffered saline (purchased from ElectronMicroscopy Sciences, #15710). Cells were imaged under on a Nikon TiEmicroscope with a 60× objective.

II. Results

FIG. 14 depicts phopshorylation of LRRK2. LRRK2 is phosphorylated onSerines 955 and 973. Mass spectrometry reports describe phosphorylationsites on LRRK2 in a cluster of serines in the amino terminus, precedingthe LRR domain. Phosphorylation of LRRK2 on Serines 910 and 935 in thiscluster of constitutive phosphorylation sites mediates an interactionwith 14-3-3 proteins and is essential for maintaining a diffusecytoplasmic localization. In order to further characterize this clusterof phosphorylation sites, HEK293 T-REx Flp-in cell lines were generatedthat express GFP tagged, full length LRRK2 and the indicated mutants ina stable and inducible manner. These lines include a GFP control, kinaseinactive LRRK2 [D2017A], phospho site mutations [Ser910A Ser935A,Ser955A, Ser973A Ser976A and combinations thereof], inhibitor resistantmutants [A2016T and A2016T+G2019S], non pathogenic PD associatedmutations [M712V, 11122V, A1442P, V1613A, E1874Stop and T2031S],pathogenic PD mutants [R1441G/C, G2019S and 12020T], and thesusceptibility factor [G2385R].

Cultures were treated with doxycycline and either treated with 1 μMLRRK2-IN1 or DMSO vehicle for 90 minutes. The TritonX-100 solublefraction was subjected to GFP nano trap immunoaffinity chromatography; arepresentative aGFP immunoblot of these immunoprecipitates is shown(aGFP, FIG. 15). To specifically detect the amino terminal phosphosites,phosphoantibodies were generated against phosphoserine 910,phosphoserine 935, phosphoserine 955 and phosphoserine 973.

To ensure specificity, antibodies were tested against the correspondingSer to Ala mutations of LRRK2. The anti-phosphoserine 910 and 935specifically recognize phosphorylated Ser910 and Ser935 (FIG. 15), nottheir Alanine mutations. The apSer955 and apSer973 antibodiesefficiently recognize phosphorylated LRRK2 on Serines 955 and 973 and donot recognize Ser955Ala or Ser973Ala mutations, confirming that thesesites are bone fide LRRK2 phosphorylation sites. Consistent with theseresults, a diphosphorylated peptide has been identified showing bothSer973 and Ser976 as phosphosites. The peptide used as an immunogendisplayed both pSer973 and pSer976, however this antibody recognizes aSer976Ala mutation as efficiently as it does wild-type LRRK2 indicatinga specificity for pSer973. When mutations in Serines 910 and 935 areanalyzed with these antibodies, no interdependence is observed withSer955 phosphorylation on these important modifications. However,mutation of Serines 910 or 935 to Alanine results in a diminution ofpSer973. LRRK2-IN1 treatment also resulted in reduced direct 14-3-3association. FIG. 16 shows an alignment of Serines 860, 910, 935, 955and 973; the primary amino acid sequence surrounding these sites revealsthat Serines 910 935 and 973 bear striking similarity except for an Aspat the −1 position at Ser973, instead of an Asn at the −1 for Ser910 andSer935. The apparent interdependence could reflect that the same or asimilar kinase modifies these residues processively, however thisremains to be tested. The direct association of 14-3-3 with LRRK2 wasassessed by 14-3-3 overlay assay using fluorescent dye labeledrecombinant 14-3-3.

Mutation of Serines 910 and 935 disrupted 14-3-3 binding and mutation ofSer955 and 973 had no effect on direct 14-3-3 binding. There was nodifference in the amounts of Hsp90 co-immunoprecipitation among thephospho-mutants, however a consistent increase in Hsp90 association withkinase inactive LRRK2 was observed and no Hsp90 was retrieved in theE1874stop mutant. These data indicate that Hsp90 associates with thekinase domain of LRRK2, and is consistent with previous reports REF.Phosphorylation of Serines 955 and 973 is regulated by LRRK2 kinaseactivity.

We evaluated the phosphorylation status of 955 and 973 under a varietyof conditions to characterize the phosphorylation of 955 and 973. Theselective kinase inhibitor of LRRK2, LRRK2-IN1, potently inhibits LRRK2in cell culture model systems and in vivo. Treatment of T-REx cellsexpressing active LRRK2 with LRRK2-IN1 results in a completedephosphorylation of Serines 910/935, as well as the dephosphorylationof Serines 955 and 973. This LRRK2-IN1 induced dephosphorylation wasobserved in all mutants that had detectable levels of phosphorylation inthe absence of inhibitor (−). Similar phosphorylation levels of 955 and973 were observed in inhibitor resistant mutants [Ala2016Thr andGly2019Ser+Ala2016Thr] regardless of LRRK2-IN1 treatment, demonstratingthat the LRRK2-IN1 effect is due to specific inhibition of LRRK2 kinaseactivity. Just as treatment with H-1152 and Sunitinib did not perturb abasal level of LRRK2 phosphorylation at 910/935 in kinase inactivemutants, no change was observed in phosphorylation of Serines910 and 935or in 955 and 973 after LRRK2-IN1 treatment of kinase inactive mutants.This low level of modification that is unperturbed by LRRK2-IN1treatment could be a basal level of phosphorylation observed when cellsare exposed to the expression of a non-natural kinase inactive mutant.Additionally, it was observed that the PD associated mutation E1874Stop,which lacks the LRRK2 kinase domain, is also not modified on Serines 955or 973. The kinase domain, regardless of the presence of inactivatingmutations, is therefore likely necessary for potentiation of feedbackphosphorylation back to the amino terminal cluster. PD mutations disruptphosphorylation of Serines 955 and 973. Pathogenic PD associatedmutations that have an increased propensity to aggregate, LRRK2[R1441C/G/H, Y1699C, 12020T], are not modified on serines 910 or 935,and therefore do not bind 14-3-3.

The impact of LRRK2 [R1441C/G, 12020T] as well as G2019S and othernon-pathogenic PD associated mutations was assessed on thephosphorylation of Serines 955 and 973. Aggregation prone/non-14-3-3binding mutations were not modified and did not show reducedmodification on Serines 955 or 973, as has been observed for Serines 910and 935. The risk factor mutation G2385R also induced reducedphosphorylation of Serines 910, 935, 955, and 973. Ser955 was moreheavily impacted by G2385R than phosphorylation of Serines 910, 935 or973.

FIG. 17 shows localization of Ser955Ala and Ser973Ala mutations.Mutation of Serines 910 and 935, individually or in combination, inducesthe accumulation of LRRK2 in the cytoplasm of HEK293 cells. Inhibitionof LRRK2 induced re-localization of the GFP tagged enzyme tointracellular puncta, large accumulations and microtubule likestructures. Localization of Ser955Ala and Ser973Ala was comparable towild-type LRRK2 localization and displayed no aggregation phenotypesimilar to 910/935. Treatment of cells expressing GFP-LRRK2Ser910Ala/Ser935Ala, Ser955Ala, and Ser973Ala with LRRK2-IN1 resulted ina similar relocalization pattern compared to wild-type LRRK2 treatedwith LRRK2-IN1. These data indicate that cytoplasmic inclusions inducedby loss of 14-3-3 binding due to Ser910Ala/Ser935Ala mutations aredistinct from those induced by the acute inhibition of LRRK2.

FIG. 18 shows phosphorylation of endogenous LRRK2. Isolation andanalysis of endogenous LRRK2 is an essential component of elucidatingthe in vivo roles of the enzyme. The first reported antibody capable ofimmunoprecipitating LRRK2 was produced in sheep against an antigencomprising amino acids 100-500 of the human enzyme. We have developedanalogous antibodies in rabbits that immunoprecipitate LRRK2. FIG. 18Ashows the ability of this reagent to isolate LRRK2 from Swiss3T3 cells.Additionally, we have characterized a mouse monoclonal antibody(NeuroMab) that is also capable of immunoprecipitating endogenous LRRK2.In order to determine if Ser955 and Ser973 are phosphorylated onendogenous protein, these antibodies were used in combination toimmunoprecipitate endogenous LRRK2 from lysates of Swiss3T3 cellstreated with LRRK2-IN1 or vehicle control. Immunoblotting theimmunoprecipitates with the apSer910, apSer935, apSer955 and apSer973antibodies reveals that endogenous LRRK2 is phosphorylated on Serines955 and 973, similar to Ser910 and Ser935.

FIG. 19 shows Characterization of anti-Thr1491 autophosphorylationantibody. In collaboration with the Michael J Fox Foundation'sAntibodies Working Group, a rabbit polyclonal antibody againstphosphorylated Ser1491 was developed. Ser1491 is known to be a site ofLRRK2 autophosphorylation. Wild-type and Ser1491Ala variants of LRRK2were transiently expressed in HEK293 cells and followingimmunoprecipitation-kinase assay, immunoprecipitates were probed withanti-phospho-Thr1491 (pThr1491) antibody. The anti-pThr1491 antibodydoes not recognize a Thr1491Ala mutant, and only reacts with LRRK2incubated in the presence of magnesium:ATP. Minimal reactivity againstpThr1491 in wild-type LRRK2 expressed in cells may indicate this site ismodified to a low stoichiometry in vivo and that immunological detectionis only capable after in vitro autophosphorylation.

LRRK2 does not phosphorylate Serines 955 and 973. The proposed feedbackphosphorylation mechanism that leads to the modification of Serines 910and 935 appears to apply to LRRK2 Ser955 and Ser973. Active LRRK2 thatwas dephosphorylated at these sites was generated and it was determinedif in vitro autophosphorylation could rephosphorylate these sites. HEK293 cells transiently expressing FLAG tagged wild-type LRRK2 weretreated with LRRK2-IN1 to dephosphorylate LRRK2, or DMSO vehiclecontrol. LRRK2 was immunoprecipitated with anti-FLAG agarose andimmunoprecipitates were washed to remove compound. Washedimmunoprecipitates were subjected to in vitro autophosphorylation kinaseassay by incubation in the presence or absence of magnesium:ATP, with orwithout LRRK2-IN1. The LRRK2 enzyme purified was able toautophosphorylate as revealed by immunoblotting with anti-pThr1491antibody. Immunoblotting with anti-pSer910, anti-pSer935, anti-pSer955and anti-pSer973 antibodies revealed that LRRK2 was indeeddephosphorylated at the corresponding residues and, importantly, thatthe phosphorylation of these sites does not increase in conditions thatare permissive for autophosphorylation (see anti-pThr1491 analysis).These data strongly argue that LRRK2 does not autophosphorylate onSerines 910, 935, 955 and 973. Cumulatively, the data indicate thatLRRK2 Serines 955 and 973 are regulated similarly to Serines 910 and935. The downstream kinase(s) and phosphatases responsible for theregulation of this constitutive phosphorylation cluster have yet to beidentified but are now targets for pathway development around LRRK2activities.

Example 28 Disruption of LRRK2 Interactions and Localization by MultipleParkinson's Disease-Associated Mutations I. Methods A. Reagents andGeneral Methods

Tissue-culture reagents were from Life Technologies. P81phosphocellulose paper was from Whatman and [γ-32P] ATP was fromPerkinElmer. All peptides were synthesized by Pepceuticals. The Flp-inT-REx system was from Invitrogen, and stable cell lines, generatedaccording to the manufacturer's instructions by selection withhygromycin. Restriction enzyme digests, DNA ligations and otherrecombinant DNA procedures were performed using standard protocols. Allmutagenesis was carried out using the QuikChange® site-directedmutagenesis kit (Stratagene). DNA constructs used for transfection werepurified from Escherichia coli DH5a cells using Qiagen or Invitrogenplasmid Maxi kits according to the manufacturer's instructions. All DNAconstructs were verified by DNA sequencing, which was performed by theSequencing Service, School of Life Sciences, University of Dundee,Dundee, Scotland, U.K., using DYEnamic ET terminator chemistry (AmershamBiosciences) with automated DNA sequencers (Applied Biosystems).

B. Buffers

Lysis buffer contained 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1%(w/v) (1 mM) sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM benzamidine and2 mM PMSF and was supplemented with 1% Triton X-100. Buffer A contained50 mM Tris/HCl, pH 7.5, 50 mM NaCl, 0.1 mM EGTA and 0.27 M sucrose. λphosphatase reactions were carried out in buffer A supplemented with 1mM MnCl2, 2 mM DTT (dithiothreitol) and 0.5 μg of λ phosphatase. MARK3(microtubule affinity-regulating kinase 3) was from UpstateBiotechnology (#05-680).

C. Cell Culture, Treatments and Cell Lysis

HEK-293 (human embryonic kidney) and Swiss 3T3 cells were cultured inDMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS(fetal bovine serum), 2 mM glutamine and 1 xantimycotic/antibioticsolution (1× penicillin/streptomycin/amphotericin B; Invitrogen).HEK-293 T-REx cell lines were cultured in DMEM supplemented with 10% FBSand 2 mM glutamine, 1× antimycotic/antibiotic solution, 15μg/mlblastocidin and 100 μg/ml hygromycin. Cell transfections wereperformed by the polyethyleneimine method. T-REx cultures were inducedto express the indicated protein by inclusion of 1 μg/ml doxycycline inthe culture medium for 24 h. Per 15 cm dish, cells washed once with PBSand lysed insitu with 1.0 ml of lysis buffer, on ice, then centrifugedat 16 000 g at 4° C. for 10 min. Protein concentrations were determinedusing the Bradford method with BSA as the standard.

D. SILAC (Stable Isotope Labelling of Amino Acids)

SILAC DMEM (high glucose without NaHCO3, L-glutamine, arginine, lysineand methionine; Biosera #A0347) was prepared with 10% dialysed FBS(Hyclone) and supplemented with methionine, glutamine, NaHCO₃ andlabelled or unlabelled arginine and lysine. Cells harbouring GFP-taggedproteins were cultured in SILAC DMEM for three passages at a 1:10 ratiowith the following isotopic labelling. For GFP compared with wild-typeLRRK2, L-arginine (84 μg/ml; Sigma-Aldrich) and L-lysine (146 μg/ml;Sigma-Aldrich) were added to the GFP ‘light’ medium, whereas13C-labelled L-arginine and 13 C-labelled

L-lysine (Cambridge Isotope Laboratory) were added to the GFP-LRRK2wild-type ‘heavy’ medium at the same concentrations. For the GFPcompared with LRRK2(G2019S) experiments, Larginineand L-lysine wereadded to the GFP ‘light’ medium and 13C/15N-labelled L-arginine and13C/15N-labelled L-lysine (Cambridge Isotope Laboratory) to theGFP-LRRK2(G2019S) ‘heavy’ medium. The amino acid concentrations arebased on the formula for normal DMEM (Invitrogen). Once prepared, theSILAC medium was mixed well and filtered through a 0.22-μm filter(Millipore). Metabolically labelled cells were induced to express GFP orthe GFP-LRRK2 fusion protein for 24 h by inclusion of doxycycline in theculture medium.

E. SILAC Labelling and MS

Cells metabolically labelled and induced to express GFP, wildtype LRRK2or LRRK2(G2019S) were lysed in lysis buffer supplemented with 1% TritonX-100 at 0.5 ml per 10 cm dish. For each condition individually, 9 mg ofcell lysate was subjected to individual immunoprecipitation with a 20 μlof bed volume of GFP-binder agarose beads for 1 h at 4° C. Beads werewashed once with 5 ml and then with 10 ml of lysis buffer supplementedwith 1% Triton-X 100 and 300 mM NaCl. Beads were then washed once with 5ml and then once with 10 ml of storage buffer. Bead-associated proteinswere eluted with 1× NuPAGE LDS sample buffer (Invitrogen) for 10 min at70° C. then passed through a 0.22 μm Spin-X column (Corning). ControlGFP eluates were combined with either eluates of wild-type LRRK2 orLRRK2(G2019S) in equal amounts and reduced and alkylated as above.Samples were resolved on a 12% Novex gel for only one half of the gel.Gels were stained with Colloidal Blue overnight and destained for 3 h.The entire lane was excised in nine bands in total and digested withtrypsin. The digests were separated on a Biosphere C18 trap column [0.1mm (internal diameter)×2 mm; Nanoseparations] connected to a PepMapC18nano column (75 μm×15 cm; Dionex Corporation) fitted to a ProxeonEasy-LC nanoflow LC-system (Proxeon Biosystems) with solvent A (2%acetonitrile/0.1% formic acid/98% water) and solvent B (90%acetonitrile/10% water/0.09% formic acid). Samples (10 μl; a total of 2μg of protein) were loaded with a constant flow of 7 μl/min on to thetrap column in solvent A and washed for 3 min at the same flow rate.After trap enrichment, peptides were eluted with a linear gradient of5-50% solvent B over 90 min with a constant flow of 300 nl/min. The HPLCsystem was coupled to a linear ion-trap-orbitrap hybrid massspectrometer (LTQ-Orbitrap XL,

Thermo Fisher Scientific) via a nanoelectrospray ion source (ProxeonBiosystems) fitted with a 5 cm Picotip FS360-20-10 emitter. The sprayvoltage was set to 1.2 kV and the temperature of the heated capillarywas set to 200° C. Full-scan MS survey spectra (m/z 350-1800) in profilemode were acquired with the LTQ-Orbitrap with a resolution of 60 000after accumulation of 500 000 ions. The five most intense peptide ionsfrom the preview scan in the LTQ-Orbitrap were fragmented bycollision-induceddissociation (normalized collision energy 35%,activation Q 0.250 and activation time 30 ms) in the LTQ-Orbitrap afterthe accumulation of 10 000 ions. Maximal filling times were 1000 ms forthe full scans and 150 ms for the MS/MS scans. Precursor ion chargestate screening was enabled and all unassigned charge states, as well assingly charged species, were rejected. The lock mass option was enabledfor survey scans to improve mass accuracy. Data were acquired using theXcalibur software.

F. LC-MS Data Analysis Using MaxQuant

The raw mass spectrometric data files obtained for each experiment werecollated into a single quantified dataset using MaxQuant (version1.0.13.13) and the Mascot search engine (Matrix Science, version 2.2.2)software. Enzyme specificity was set to that of trypsin. Otherparameters used within the software: variable modifications, methionineoxidation; database, target-decoy human MaxQuant (ipi.HUMAN.v3.52.decoy)(containing 148,380 database entries); labels, R6K4 (for GFP comparedwith wild-type LRRK2) or R10K8 [for GFP compared with LRRK2(G2019S)];MS/MS tolerance, 0.5 Da; top MS/MS peaks per 100 Da, 5; maximum missedcleavages, 2; maximum of labelled amino acids, 3; FDR (false discoveryrate), 1%.

G. Phosphorylation Site Identification by MS

Endogenous and recombinant LRRK2 was immunoprecipitated from 50 mg ofSwiss 3T3 lysate or T-REx cells induced to express FLAG-LRRK2 celllysate using anti-LRRK2-(100-500)—or anti-FLAG-agarose respectively.Immunoprecipitates were eluted from the affinity matrices using 2×LDSsample buffer or 200 μg/ml FLAG peptide then filtered through a 0.2 μmSpin-X column before reduction with 10 mM DTT and alkylation with 50 mMiodoacetamide. Samples were heated for 10 min at 70° C. and resolved on4-12% Novex gels before staining with Colloidal Blue (Invitrogen). Bandscorresponding to LRRK2 were excised and digested with trypsin asdescribed previously. Samples were analysed on an LTQ-Orbitrap XL massspectrometer as described above, except that the top five ions werefragmented in the linear ion-trap using multistage activation of theneutral loss of phosphoric acid from the parent ion (neutral lossmasses=49,32.33 and 24.5 for z=2, 3 and 4 respectively). Mascot genericfiles were created from the raw files using raw2msm (a gift fromProfessor Matthias Mann, Max Planck Institute of Biochemistry,Martinsried, Germany) and were searched on a local Mascot server usingthe IPI (International Protein Index) mouse database for endogenousLRRK2 or the IPI human database for recombinant LRRK2.

H. Immunological Procedures

Cell lysates (10-30 μg) were resolved by electrophoresis onSDS/polyacrylamide gels or Novex 4-12% gradient gels, and electroblottedon to nitrocellulose membranes. Membranes were blocked with 5% (w/v)skimmed milk in TBST [Tris-buffered saline with Tween 20: 50 mMTris/HCl, pH 7.5, 0.15 M NaCl and 0.1% (v/v) Tween 20]. Forphospho-specific antibodies, primary antibody was used at aconcentration of 1 μg/ml, diluted in 5% skimmed milk in TBST with theinclusion of 10 μg/ml dephosphorylated peptide. All other antibodieswere used at 1 μg/ml in 5% (w/v) milk in TBST. Detection ofimmuno-complexes was performed using either fluorophoreconjugatedsecondary antibodies (Molecular Probes) followed by visualization usingan Odyssey infrared imaging system (LI-COR Biosciences) or byhorseradish-peroxidase-conjugated secondary antibodies (Pierce) and anenhanced chemiluminescence reagent. For immunoprecipitations, antibodywas non-covalently coupled to Protein G-Sepharose at a ratio of 1 μg ofantibody/μl of beads, or anti-FLAG M2-agarose was utilized. Cell lysateswere incubated with coupled antibody for 1 h. To assess Ser935phosphorylation, total LRRK2 levels and 14-3-3 binding in mouse tissues,LRRK2 was immunoprecipitated from 6 mg of wholetissue lysate using 15 μgof antibody coupled to 15 μl of Protein G-Sepharose. Ser910phosphorylation was assessed following immunoprecipitation from 10 mg oftissue lysate. Immunocomplexes were washed twice with lysis buffersupplemented with 0.3 M NaCl and twice with buffer A. Precipitates werere-suspended in LDS sample buffer and subjected to immunoblot analysis.DIG (digoxigenin)-labelled 14-3-3 for use in overlay far-Western blotanalysis. To directly assess 14-3-3 interaction with LRRK2,immunoprecipitates were electroblotted on to nitrocellulose membranesand blocked with 5% skimmed milk for 30 min. After washing with TBST,membranes were incubated with DIG-labelled 14-3-3 diluted to 1 μg/ml in5% BSA in TBST overnight at 4° C. DIG-labelled 14-3-3 was detected withhorseradishperoxidase-labelled anti-DIG Fab fragments (Roche).

I. LRRK2 Immunoprecipitation Kinase Assays

Transfected cell lysates (500 μg) were subjected to immunoprecipitationwith a 5 μl bed volume of anti-FLAG-agarose for 1 h. Beads were washedtwice with lysis buffer supplemented with 300 mM NaCl, and twice withbuffer A. Peptide kinase assay were set up in a total volume of 50 μlwith immunoprecipitated LRRK2 in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10mM MgCl2 and 0.1 mM [γ-32P]ATP (˜300-500 c.p.m./pmol) in the presence of200 μM long variant of the LRRKtide peptide substrate(RLGRDKYKTLRQIRQGNTKQR (SEQ ID NO: 5)) or the Nictide peptide substrate(RLGWWRFYTLRRARQGNTKQR (SEQ ID NO: 6)). Reactions were terminated byapplying 30 μl of the reaction mixture on to P81 phosphocellulose paperand immersing in 50 mM phosphoric acid. After extensive washing, theradioactivity in the reaction products was quantified by Cerenkovcounting. One half of the remaining reaction was subjected to immunoblotanalysis using the Odyssey infrared imaging system and specific activityis represented as c.p.m./independent density values. Affinitypurification of 14-3-3 with a di-phosphorylated peptide encompassingSer910 and Ser935 An N-terminally biotinylated di-phosphorylated peptideencompassing Ser910 and Ser935(biotin-KKKSNpSISVGEFYRDAVLQRCSPNLQRHSNpSLGPIF (SEQ ID NO: 7)) wasconjugated to streptavidin-agarose (1 μg peptide/pg of agarose).Aliquots of agarose beads (10 μl) were treated with or without λphosphatase for 30 min at 30° C., with λ phosphatase being in thepresence or absence of 50 mM EDTA. Conjugated beads were then incubatedwith 3 mg of HEK-293 cell lysate at 4° C. for 1 h. Following two washeswith lysis buffer supplemented with 0.5 M NaCl, beads were boiled in LDSsample buffer and samples subjected to immunoblot analysis for 14-3-3.

FIG. 21 shows that phosphorylation of Serines 955 and 973 is Dependenton LRRK2 Kinase Activity.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of editing an LRRK2 gene comprising: a)contacting the LRRK2 gene with an engineered nuclease that cleaves theLRRK2 gene at a site in the LRRK2 gene; and b) producing a basealteration, base deletion or base insertion at the site that results inan amino acid change in an LRRK2 protein encoded by the LRRK2 gene. 2.The method of claim 1, wherein the amino acid change results in reducedaggregation of LRRK2 proteins encoded by the LRRK2 gene.
 3. The methodof claim 1, wherein the amino acid change results in altering a kinaseactivity of the LRRK2 protein.
 4. The method of claim 1, wherein theamino acid change occurs at a position in the LRRK2 protein selectedfrom amino acid positions 910, 935, 955, 973, 1441, 1699, 2019, and 2020of the LRRK2 protein.
 5. The method of claim 1, wherein producing theamino acid change restores a phosphorylation site in the LRRK2 protein.6. The method of claim 1, wherein the editing occurs in a neuronal stemcell.
 7. The method of claim 1, wherein the editing occurs in an inducedpluripotent stem cell.
 8. The method of claim 1, wherein the engineerednuclease produces a double-stranded break in the LRRK2 gene.
 9. Themethod of claim 8, wherein the double stranded break is resolved throughhomology-directed repair or non-homologous end joining.
 10. The methodof claim 8, wherein the double stranded break is repaired with a donorDNA template.
 11. The method of claim 1, wherein the engineered nucleaseis a zinc finger nuclease.
 12. The method of claim 1, wherein the basealteration, base deletion or base insertion results in correcting amutation in the LRRK2 protein selected from R1441C, R1441G, Y1699C,12020T, S910A, S910E, S910D, S955A, S955E, S955D, S973A, S973E, andS973D.
 13. A cell comprising an LRRK2 gene modified by an engineerednuclease.
 14. The cell of claim 13, wherein the cell is a neuronal stemcell.
 15. The cell of claim 13, wherein the cells is an inducedpluripotent stem cell.
 16. The cell of claim 13, wherein the LRRK2 geneis modified to correct a genetic mutation, wherein the mutation causesan amino acid alteration in an LRRK2 protein encoded by the LRRK2 geneselected from R1441C, R1441G, Y1699C, 12020T, S910A, S910E, S910D,S955A, S955E, S955D, S973A, S973E, and S973D.
 17. The cell of claim 13,wherein the LRRK2 protein comprised a mutation before being modified bythe engineered nuclease, and wherein the mutation is selected fromR1441C, R1441G, Y1699C, 12020T, S910A, S910E, S910D, S955A, S955E,S955D, S973A, S973E, and S973D.
 18. A method of treating a subject for acondition comprising administering the cell of claim 13 to the subject.19. The method of claim 18, wherein the condition is a neurodegenerativedisease.
 20. The method of claim 18, wherein the condition isParkinson's disease.